Nancy C Horton

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Scholarly Contributions

Chapters

• Horton, N., & Park, C. (2010). Crystallization of Zinc Finger Proteins bound to DNA. In Methods Mol Biol(pp 457-477).
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  Volume: 649
• Horton, N., & Park, C. (2007). Deoxyribonucleases. In Protein-Nucleic Acid Interactions: Structural Biology. Cambridge, United Kingdom: RSC Publishing.
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  Book editors: Correll, CC | Rice, P; 2008

Journals/Publications

• Ghadirian, N., Morgan, R. D., & Horton, N. C. (2024). DNA Sequence Control of Enzyme Filamentation and Activation of the SgrAI Endonuclease. Biochemistry, 63(3), 326-338.
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  Enzyme polymerization (also known as filamentation) has emerged as a new layer of enzyme regulation. SgrAI is a sequence-dependent DNA endonuclease that forms polymeric filaments with enhanced DNA cleavage activity as well as altered DNA sequence specificity. To better understand this unusual regulatory mechanism, full global kinetic modeling of the reaction pathway, including the enzyme filamentation steps, has been undertaken. Prior work with the primary DNA recognition sequence cleaved by SgrAI has shown how the kinetic rate constants of each reaction step are tuned to maximize activation and DNA cleavage while minimizing the extent of DNA cleavage to the host genome. In the current work, we expand on our prior study by now including DNA cleavage of a secondary recognition sequence, to understand how the sequence of the bound DNA modulates filamentation and activation of SgrAI. The work shows that an allosteric equilibrium between low and high activity states is modulated by the sequence of bound DNA, with primary sequences more prone to activation and filament formation, while SgrAI bound to secondary recognition sequences favor the low (and nonfilamenting) state by up to 40-fold. In addition, the degree of methylation of secondary sequences in the host organism, , is now reported for the first time and shows that as predicted, these sequences are left unprotected from the SgrAI endonuclease making sequence specificity critical in this unusual filament-forming enzyme.
• Zerio, C. J., Sivinski, J., Wijeratne, E. M., Xu, Y. M., Ngo, D. T., Ambrose, A. J., Villa-Celis, L., Ghadirian, N., Clarkson, M. W., Zhang, D. D., Horton, N. C., Gunatilaka, A. A., Fromme, R., & Chapman, E. (2023). Physachenolide C is a Potent, Selective BET Inhibitor. Journal of medicinal chemistry, 66(1), 913-933.
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  A pulldown using a biotinylated natural product of interest in the 17β-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), identified the bromodomain and extra-terminal domain (BET) family of proteins (BRD2, BRD3, and BRD4), readers of acetyl-lysine modifications and regulators of gene transcription, as potential cellular targets. BROMOscan bromodomain profiling and biochemical assays support PCC as a BET inhibitor with increased selectivity for bromodomain (BD)-1 of BRD3 and BRD4, and X-ray crystallography and NMR studies uncovered specific contacts that underlie the potency and selectivity of PCC toward BRD3-BD1 over BRD3-BD2. PCC also displays characteristics of a molecular glue, facilitating proteasome-mediated degradation of BRD3 and BRD4. Finally, PCC is more potent than other withanolide analogues and gold-standard pan-BET inhibitor (+)-JQ1 in cytotoxicity assays across five prostate cancer (PC) cell lines regardless of androgen receptor (AR)-signaling status.
• Lyumkis, D., & Horton, N. C. (2022). The role of filamentation in activation and DNA sequence specificity of the sequence-specific endonuclease SgrAI. Biochemical Society transactions, 50(6), 1703-1714.
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  Filament formation by metabolic, biosynthetic, and other enzymes has recently come into focus as a mechanism to fine-tune enzyme activity in the cell. Filamentation is key to the function of SgrAI, a sequence-specific DNA endonuclease that has served as a model system to provide some of the deepest insights into the biophysical characteristics of filamentation and its functional consequences. Structure-function analyses reveal that, in the filamentous state, SgrAI stabilizes an activated enzyme conformation that leads to accelerated DNA cleavage activity and expanded DNA sequence specificity. The latter is thought to be mediated by sequence-specific DNA structure, protein-DNA interactions, and a disorder-to-order transition in the protein, which collectively affect the relative stabilities of the inactive, non-filamentous conformation and the active, filamentous conformation of SgrAI bound to DNA. Full global kinetic modeling of the DNA cleavage pathway reveals a slow, rate-limiting, second-order association rate constant for filament assembly, and simulations of in vivo activity predict that filamentation is superior to non-filamenting mechanisms in ensuring rapid activation and sequestration of SgrAI's DNA cleavage activity on phage DNA and away from the host chromosome. In vivo studies demonstrate the critical requirement for accelerated DNA cleavage by SgrAI in its biological role to safeguard the bacterial host. Collectively, these data have advanced our understanding of how filamentation can regulate enzyme structure and function, while the experimental strategies used for SgrAI can be applied to other enzymatic systems to identify novel functional roles for filamentation.
• Sanchez, J. L., Ghadirian, N., & Horton, N. C. (2022). High-Resolution Structure of the Nuclease Domain of the Human Parvovirus B19 Main Replication Protein NS1. Journal of virology, 96(9), e0216421.
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  Two new structures of the N-terminal domain of the main replication protein, NS1, of human parvovirus B19 (B19V) are presented here. This domain (NS1-nuc) plays an important role in the "rolling hairpin" replication of the single-stranded B19V DNA genome, recognizing origin of replication sequences in double-stranded DNA, and cleaving (i.e., nicking) single-stranded DNA at a nearby site known as the terminal resolution site (trs). The three-dimensional structure of NS1-nuc is well conserved between the two forms, as well as with a previously solved structure of a sequence variant of the same domain; however, it is shown here at a significantly higher resolution (2.4 Å). Using structures of NS1-nuc homologues bound to single- and double-stranded DNA, models for DNA recognition and nicking by B19V NS1-nuc are presented that predict residues important for DNA cleavage and for sequence-specific recognition at the viral origin of replication. The high-resolution structure of the DNA binding and cleavage domain of the main replicative protein, NS1, from the human-pathogenic virus human parvovirus B19 is presented here. Included also are predictions of how the protein recognizes important sequences in the viral DNA which are required for viral replication. These predictions can be used to further investigate the function of this protein, as well as to predict the effects on viral viability due to mutations in the viral protein and viral DNA sequences. Finally, the high-resolution structure facilitates structure-guided drug design efforts to develop antiviral compounds against this important human pathogen.
• Shan, Z., Ghadirian, N., Lyumkis, D., & Horton, N. C. (2022). Pretransition state and apo structures of the filament-forming enzyme SgrAI elucidate mechanisms of activation and substrate specificity. The Journal of biological chemistry, 298(4), 101760.
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  Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both accelerates its DNA cleavage activity and expands its DNA sequence specificity, thus allowing for many additional DNA sequences to be rapidly cleaved. Both outcomes-the acceleration of DNA cleavage and the expansion of sequence specificity-are proposed to regulate critical processes in bacterial innate immunity. However, the mechanistic bases underlying these events remain unclear. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca resolved to ∼2.5 Å within the catalytic center, which represents the trapped enzyme-DNA complex prior to the DNA cleavage reaction. This structure reveals important conformational changes that contribute to the catalytic mechanism and the binding of a second divalent cation in the enzyme active site, which is expected to contribute to increased DNA cleavage activity of SgrAI in the filamentous state. Second, we present an X-ray crystal structure of DNA-free (apo) SgrAI resolved to 2.0 Å resolution, which reveals a disordered loop involved in DNA recognition. Collectively, these multiple new observations clarify the mechanism of expansion of DNA sequence specificity of SgrAI, including the indirect readout of sequence-dependent DNA structure, changes in protein-DNA interactions, and the disorder-to-order transition of a crucial DNA recognition element.
• Townsend, J. A., Sanders, H. M., Rolland, A. D., Park, C. K., Horton, N. C., Prell, J. S., Wang, J., & Marty, M. T. (2021). Influenza AM2 Channel Oligomerization Is Sensitive to Its Chemical Environment. Analytical chemistry, 93(48), 16273-16281.
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  Viroporins are small viral ion channels that play important roles in the viral infection cycle and are proven antiviral drug targets. Matrix protein 2 from influenza A (AM2) is the best-characterized viroporin, and the current paradigm is that AM2 forms monodisperse tetramers. Here, we used native mass spectrometry and other techniques to characterize the oligomeric state of both the full-length and transmembrane (TM) domain of AM2 in a variety of different pH and detergent conditions. Unexpectedly, we discovered that AM2 formed a range of different oligomeric complexes that were strongly influenced by the local chemical environment. Native mass spectrometry of AM2 in nanodiscs with different lipids showed that lipids also affected the oligomeric states of AM2. Finally, nanodiscs uniquely enabled the measurement of amantadine binding stoichiometries to AM2 in the intact lipid bilayer. These unexpected results reveal that AM2 can form a wider range of oligomeric states than previously thought possible, which may provide new potential mechanisms of influenza pathology and pharmacology.
• Park, C. K., & Horton, N. C. (2020). Novel insights into filament-forming enzymes. Nature reviews. Molecular cell biology, 21(1), 1-2.
• Park, C. K., & Horton, N. C. (2019). Structures, functions, and mechanisms of filament forming enzymes: a renaissance of enzyme filamentation. Biophysical reviews, 11(6), 927-994.
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  Filament formation by non-cytoskeletal enzymes has been known for decades, yet only relatively recently has its wide-spread role in enzyme regulation and biology come to be appreciated. This comprehensive review summarizes what is known for each enzyme confirmed to form filamentous structures in vitro, and for the many that are known only to form large self-assemblies within cells. For some enzymes, studies describing both the in vitro filamentous structures and cellular self-assembly formation are also known and described. Special attention is paid to the detailed structures of each type of enzyme filament, as well as the roles the structures play in enzyme regulation and in biology. Where it is known or hypothesized, the advantages conferred by enzyme filamentation are reviewed. Finally, the similarities, differences, and comparison to the SgrAI endonuclease system are also highlighted.
• Polley, S., Lyumkis, D., & Horton, N. C. (2019). Mechanism of Filamentation-Induced Allosteric Activation of the SgrAI Endonuclease. Structure (London, England : 1993), 27(10), 1497-1507.e3.
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  Filament formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the cryoelectron microscopy (cryo-EM) structure of the filament of SgrAI in its activated configuration. The structural data illuminate the mechanistic origin of hyperaccelerated DNA cleavage activity and suggests how indirect DNA sequence readout within filamentous SgrAI may enable recognition of substantially more nucleotide sequences than its low-activity form, thereby altering and partially relaxing its DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAI's catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.
• Barahona, C., Basantes, L. E., Tompkins, K. J., Heitman, D. M., Chukwu, B. I., Sanchez, J., Sanchez, J. L., Ghadirian, N., Park, C. K., & Horton, N. C. (2018). The Need for Speed: Run-On Oligomer Filament Formation Provides Maximum Speed with Maximum Sequestration of Activity. Journal of Virology.
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  Herein we investigate an unusual anti-viral mechanism developed in the bacterium SgrAI is a type II restriction endonuclease which forms run-on oligomer filaments when activated, and which possesses both accelerated DNA cleavage activity and expanded DNA sequence specificity. Mutations disrupting the run-on oligomer filament eliminate the robust anti-phage activity of wild type SgrAI, and the observation that even relatively modest disruptions completely abolish this anti-viral activity shows that the greater speed imparted by the run-on oligomer filament mechanism is critical to its biological function. Simulations of DNA cleavage by SgrAI uncover the origins of the kinetic advantage of this newly described mechanism of enzyme regulation over more conventional mechanisms, as well as the origin of the sequestering effect responsible for the protection of the host genome against the damaging DNA cleavage activity of activated SgrAI. This work is motivated by the interest in understanding the characteristics and advantages of a relatively newly discovered enzyme mechanism involving filament formation. SgrAI is an enzyme responsible for protecting against viral infections in its host bacterium, and was one of the first such enzymes shown to utilize such a mechanism. In this work, filament formation by SgrAI is disrupted and the effects on the speed of the purified enzyme as well as its function in cells are measured. It was found that even small disruptions, which weaken but do not destroy filament formation, eliminate the ability of SgrAI to protect cells from viral infection, its normal biological function. Simulations of enzyme activity were also performed and show how filament formation can greatly speed up an enzyme's activation compared to other known mechanisms, as well as better localize its action to molecules of interest such as invading phage DNA.
• Horton, N., Horton, N., Park, C., Sanchez, J., Barahona, C., & Basantes, E. (2018). Enzyme Activity and Specificity Modulated by Protein Filamentation. PROTEIN SCIENCE, 27, 117-118.
• Park, C. K., Sanchez, J. L., Barahona, C., Basantes, L. E., Sanchez, J., Hernandez, C., & Horton, N. C. (2018). The run-on oligomer filament enzyme mechanism of SgrAI: Part 1. Assembly kinetics of the run-on oligomer filament. The Journal of biological chemistry, 293(38), 14585-14598.
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  Filament or run-on oligomer formation by metabolic enzymes is now recognized as a widespread phenomenon having potentially unique enzyme regulatory properties and biological roles, and its dysfunction is implicated in human diseases such as cancer, diabetes, and developmental disorders. SgrAI is a bacterial allosteric type II restriction endonuclease that binds to invading phage DNA, may protect the host DNA from off-target cleavage activity, and forms run-on oligomeric filaments with enhanced DNA-cleavage activity and altered DNA sequence specificity. However, the mechanisms of SgrAI filament growth, cooperativity in filament formation, sequestration of enzyme activity, and advantages over other filament mechanisms remain unknown. In this first of a two-part series, we developed methods and models to derive association and dissociation rate constants of DNA-bound SgrAI in run-on oligomers and addressed the specific questions of cooperativity and filament growth mechanisms. We show that the derived rate constants are consistent with the run-on oligomer sizes determined by EM analysis and are most consistent with a noncooperative growth mode of the run-on oligomer. These models and methods are extended in the accompanying article to include the full DNA-cleavage pathway and address specific questions related to the run-on oligomer mechanism including the sequestration of DNA-cleavage activity and trapping of products.
• Park, C. K., Sanchez, J. L., Barahona, C., Basantes, L. E., Sanchez, J., Hernandez, C., & Horton, N. C. (2018). The run-on oligomer filament enzyme mechanism of SgrAI: Part 2. Kinetic modeling of the full DNA cleavage pathway. The Journal of biological chemistry, 293(38), 14599-14615.
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  Filament or run-on oligomer formation by enzymes is now recognized as a widespread phenomenon with potentially unique enzyme regulatory properties and biological roles. SgrAI is an allosteric type II restriction endonuclease that forms run-on oligomeric filaments with activated DNA cleavage activity and altered DNA sequence specificity. In this two-part work, we measure individual steps in the run-on oligomer filament mechanism to address specific questions of cooperativity, trapping, filament growth mechanisms, and sequestration of activity using fluorophore-labeled DNA, kinetic FRET measurements, and reaction modeling with global data fitting. The final models and rate constants show that the assembly step involving association of SgrAI-DNA complexes into the run-on oligomer filament is relatively slow (3-4 orders of magnitude slower than diffusion limited) and rate-limiting at low to moderate concentrations of SgrAI-DNA. The disassembly step involving dissociation of complexes of SgrAI-DNA from each other in the run-on oligomer filament is the next slowest step but is fast enough to limit the residence time of any one copy of SgrAI or DNA within the dynamic filament. Further, the rate constant for DNA cleavage is found to be 4 orders of magnitude faster in the run-on oligomer filament than in isolated SgrAI-DNA complexes and faster than dissociation of SgrAI-DNA complexes from the run-on oligomer filament, making the reaction efficient in that each association into the filament likely leads to DNA cleavage before filament dissociation.
• Xu, P., Ganaie, S. S., Wang, X., Wang, Z., Kleiboeker, S., Horton, N. C., Heier, R. F., Meyers, M. J., Tavis, J. E., & Qiu, J. (2018). Endonuclease Activity Inhibition of the NS1 Protein of Parvovirus B19 as a Novel Target for Antiviral Drug Development. Antimicrobial agents and chemotherapy.
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  Human parvovirus B19 (B19V), a member of the genus of the family , is a small non-enveloped virus that has a single-stranded DNA (ssDNA) genome of 5.6 kilobases with two inverted terminal repeats (ITRs). B19V infection often results in severe hematological disorders and fetal death in humans. B19V replication follows a model of rolling hairpin-dependent DNA replication, in which the large non-structural protein NS1 introduces a site-specific single strand nick in the viral DNA replication origins, which locate at the ITRs. NS1 executes endonuclease activity through the N-terminal origin binding domain. Nicking of the viral replication origin is a pivotal step in rolling hairpin-dependent viral DNA replication. Here, we developed a fluorophore-based nicking assay of the replication origin using the origin binding domain of the NS1 and compared it with the radioactive nicking assay. We used both assays to screen a set of small molecule compounds (96) that have potential anti-nuclease activity. We found that the fluorophore-based nicking assay demonstrate sensitivity and specificity values as high as the radioactive assay. Among the 96 compounds, we identified 8 which have an inhibition of >80% at 10 µM in both the fluorophore-based and radioactive nicking assays. We further tested 3 compounds that have flavonoid-like structure for IC that falls in the range of 1-3 µM. Importantly, they also exhibited inhibition of B19V DNA replication in UT7/Epo-S1 cells and expanded human erythroid progenitor cells.
• Sanchez, J. L., Romero, Z., Quinones, A., Torgeson, K. R., & Horton, N. C. (2016). DNA Binding and Cleavage by the Human Parvovirus B19 NS1 Nuclease Domain. Biochemistry, 55(47), 6577-6593.
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  Infection with human parvovirus B19 (B19V) has been associated with a myriad of illnesses, including erythema infectiosum (Fifth disease), hydrops fetalis, arthropathy, hepatitis, and cardiomyopathy, and also possibly the triggering of any number of different autoimmune diseases. B19V NS1 is a multidomain protein that plays a critical role in viral replication, with predicted nuclease, helicase, and gene transactivation activities. Herein, we investigate the biochemical activities of the nuclease domain (residues 2-176) of B19V NS1 (NS1-nuc) in sequence-specific DNA binding of the viral origin of replication sequences, as well as those of promoter sequences, including the viral p6 and the human p21, TNFα, and IL-6 promoters previously identified in NS1-dependent transcriptional transactivation. NS1-nuc was found to bind with high cooperativity and with multiple (five to seven) copies to the NS1 binding elements (NSBE) found in the viral origin of replication and the overlapping viral p6 promoter DNA sequence. NS1-nuc was also found to bind cooperatively with at least three copies to the GC-rich Sp1 binding sites of the human p21 gene promoter. Only weak or nonspecific binding of NS1-nuc to the segments of the TNFα and IL-6 promoters was found. Cleavage of DNA by NS1-nuc occurred at the expected viral sequence (the terminal resolution site), but only in single-stranded DNA, and NS1-nuc was found to covalently attach to the 5' end of the DNA at the cleavage site. Off-target cleavage by NS1-nuc was also identified.
• Shah, S., Dunten, P., Stiteler, A., Park, C. K., & Horton, N. C. (2015). Structure and specificity of FEN-1 from Methanopyrus kandleri. Proteins, 83(1), 188-94.
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  DNA repair is fundamental to genome stability and is found in all three domains of life. However many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologs, and those present often contain significant differences compared to their eukaryotic homologs. To clarify the role of the NER XPG-like protein Mk0566 from M. kandleri, its biochemical activity and three-dimensional structure were investigated. Both were found to be more similar to human FEN-1 than human XPG, suggesting a biological role in replication and long-patch base excision repair rather than in NER.
• Shah, S., Sanchez, J., Stewart, A., Piperakis, M. M., Cosstick, R., Nichols, C., Park, C. K., Ma, X., Wysocki, V., Bitinaite, J., & Horton, N. C. (2015). Probing the run-on oligomer of activated SgrAI bound to DNA. PloS one, 10(4), e0124783.
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  SgrAI is a type II restriction endonuclease with an unusual mechanism of activation involving run-on oligomerization. The run-on oligomer is formed from complexes of SgrAI bound to DNA containing its 8 bp primary recognition sequence (uncleaved or cleaved), and also binds (and thereby activates for DNA cleavage) complexes of SgrAI bound to secondary site DNA sequences which contain a single base substitution in either the 1st/8th or the 2nd/7th position of the primary recognition sequence. This modulation of enzyme activity via run-on oligomerization is a newly appreciated phenomenon that has been shown for a small but increasing number of enzymes. One outstanding question regarding the mechanistic model for SgrAI is whether or not the activating primary site DNA must be cleaved by SgrAI prior to inducing activation. Herein we show that an uncleavable primary site DNA containing a 3'-S-phosphorothiolate is in fact able to induce activation. In addition, we now show that cleavage of secondary site DNA can be activated to nearly the same degree as primary, provided a sufficient number of flanking base pairs are present. We also show differences in activation and cleavage of the two types of secondary site, and that effects of selected single site substitutions in SgrAI, as well as measured collisional cross-sections from previous work, are consistent with the cryo-electron microscopy model for the run-on activated oligomer of SgrAI bound to DNA.
• Horton, N., Ma, X., Shah, S., Zhou, M., Park, C. K., Wysocki, V. H., & Horton, N. C. (2013). Structural analysis of activated SgrAI-DNA oligomers using ion mobility mass spectrometry. Biochemistry, 52(25).
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  SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits self-modulation of DNA cleavage activity and sequence specificity. Previous studies have shown that SgrAI forms large oligomers when bound to particular DNA sequences and under the same conditions where SgrAI exhibits accelerated DNA cleavage kinetics. However, the detailed structure and stoichiometry of the SgrAI-DNA complex as well as the basic building block of the oligomers have not been fully characterized. Ion mobility mass spectrometry (IM-MS) was employed to analyze SgrAI-DNA complexes and show that the basic building block of the oligomers is the DNA-bound SgrAI dimer (DBD) with one SgrAI dimer bound to two precleaved duplex DNA molecules each containing one-half of the SgrAI primary recognition sequence. The oligomers contain variable numbers of DBDs with as many as 19 DBDs. Observation of the large oligomers shows that nanoelectrospray ionization (nano-ESI) can preserve the proposed activated form of an enzyme. Finally, the collision cross section of the SgrAI-DNA oligomers measured by IM-MS was found to have a linear relationship with the number of DBDs in each oligomer, suggesting a regular, repeating structure.
• Lyumkis, D., Talley, H., Stewart, A., Shah, S., Park, C. K., Tama, F., Potter, C. S., Carragher, B., & Horton, N. C. (2013). Allosteric regulation of DNA cleavage and sequence-specificity through run-on oligomerization. Structure (London, England : 1993), 21(10), 1848-58.
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  SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200- to 500-fold by effector DNA, with a concomitant expansion of its DNA sequence specificity. Using single-particle transmission electron microscopy to reconstruct distinct populations of SgrAI oligomers, we show that in the presence of allosteric, activating DNA, the enzyme forms regular, repeating helical structures characterized by the addition of DNA-binding dimeric SgrAI subunits in a run-on manner. We also present the structure of oligomeric SgrAI at 8.6 Å resolution, demonstrating the conformational state of SgrAI in its activated form. Activated and oligomeric SgrAI displays key protein-protein interactions near the helix axis between its N termini, as well as allosteric protein-DNA interactions that are required for enzymatic activation. The hybrid approach reveals an unusual mechanism of enzyme activation that explains SgrAI's oligomerization and allosteric behavior.
• Horton, N., Little, E. J., Dunten, P. W., Bitinaite, J., & Horton, N. C. (2011). New clues in the allosteric activation of DNA cleavage by SgrAI: structures of SgrAI bound to cleaved primary-site DNA and uncleaved secondary-site DNA. Acta crystallographica. Section D, Biological crystallography, 67(Pt 1).
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  SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-activation with expansion of DNA-sequence specificity. The three-dimensional crystal structures of SgrAI bound to cleaved primary-site DNA and Mg²(+) and bound to secondary-site DNA with either Mg²(+) or Ca²(+) are presented. All three structures show a conformation of enzyme and DNA similar to the previously determined dimeric structure of SgrAI bound to uncleaved primary-site DNA and Ca²(+) [Dunten et al. (2008), Nucleic Acids Res. 36, 5405-5416], with the exception of the cleaved bond and a slight shifting of the DNA in the SgrAI/cleaved primary-site DNA/Mg²(+) structure. In addition, a new metal ion binding site is located in one of the two active sites in this structure, which is consistent with proposals for the existence of a metal-ion site near the 3'-O leaving group.
• Horton, N. C., & Park, C. K. (2010). Crystallization of zinc finger proteins bound to DNA. Methods in molecular biology (Clifton, N.J.), 649, 457-77.
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  A method is presented for determining conditions for the cocrystallization of zinc finger proteins with DNA. The method describes steps beginning with protein expression, through purification, design of DNA for cocrystallization, and the conditions to screen for cocrystallization.
• Horton, N., Park, C. K., Joshi, H. K., Agrawal, A., Ghare, M. I., Little, E. J., Dunten, P. W., Bitinaite, J., & Horton, N. C. (2010). Domain swapping in allosteric modulation of DNA specificity. PLoS biology, 8(12).
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  SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of cleavage activity and sequence specificity. Previous studies have shown that DNA bound dimers of SgrAI oligomerize into an activated form with higher DNA cleavage rates, although previously determined crystal structures of SgrAI bound to DNA show only the DNA bound dimer. A new crystal structure of the type II restriction endonuclease SgrAI bound to DNA and Ca(2+) is now presented, which shows the close association of two DNA bound SgrAI dimers. This tetrameric form is unlike those of the homologous enzymes Cfr10I and NgoMIV and is formed by the swapping of the amino-terminal 24 amino acid residues. Two mutations predicted to destabilize the swapped form of SgrAI, P27W and P27G, have been made and shown to eliminate both the oligomerization of the DNA bound SgrAI dimers as well as the allosteric stimulation of DNA cleavage by SgrAI. A mechanism involving domain swapping is proposed to explain the unusual allosteric properties of SgrAI via association of the domain swapped tetramer of SgrAI bound to DNA into higher order oligomers.
• Horton, N., Park, C. K., Stiteler, A. P., Shah, S., Ghare, M. I., Bitinaite, J., & Horton, N. C. (2010). Activation of DNA cleavage by oligomerization of DNA-bound SgrAI. Biochemistry, 49(41).
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  SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of DNA activity and sequence specificity. Precleaved primary site DNA has been shown to be an allosteric effector [Hingorani-Varma, K., and Bitinaite, J. (2003) J. Biol. Chem. 278, 40392-40399], stimulating cleavage of both primary (CR|CCGGYG, where the vertical bar indicates a cut site, R denotes A or G, and Y denotes C or T) and secondary [CR|CCGGY(A/C/T) and CR|CCGGGG] site DNA sequences. The fact that DNA is the allosteric effector of this endonuclease suggests at least two DNA binding sites on the functional SgrAI molecule, yet crystal structures of SgrAI [Dunten, P. W., et al. (2008) Nucleic Acids Res. 36, 5405-5416] show only one DNA duplex bound to one dimer of SgrAI. We show that SgrAI forms species larger than dimers or tetramers [high-molecular weight species (HMWS)] in the presence of sufficient concentrations of SgrAI and its primary site DNA sequence that are dependent on the concentration of the DNA-bound SgrAI dimer. Analytical ultracentrifugation indicates that the HMWS is heterogeneous, has sedimentation coefficients of 15-20 s, and is composed of possibly 4-12 DNA-bound SgrAI dimers. SgrAI bound to secondary site DNA will not form HMWS itself but can bind to HMWS formed with primary site DNA and SgrAI. Uncleaved, as well as precleaved, primary site DNA is capable of stimulating HMWS formation. Stimulation of DNA cleavage by SgrAI, at primary as well as secondary sites, is also dependent on the concentration of primary site DNA (cleaved or uncleaved) bound SgrAI dimers. SgrAI bound to secondary site DNA does not have significant stimulatory activity. We propose that the oligomers of DNA-bound SgrAI (i.e., HMWS) are the activated, or activatable, forms of the enzyme.
• Dunten, P. W., Little, E. J., & Horton, N. C. (2009). The restriction enzyme SgrAI: structure solution via combination of poor MIRAS and MR phases. Acta crystallographica. Section D, Biological crystallography, 65(Pt 4), 393-8.
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  Uninterpretable electron-density maps were obtained using either MIRAS phases or MR phases in attempts to determine the structure of the type II restriction endonuclease SgrAI bound to DNA. While neither solution strategy was particularly promising (map correlation coefficients of 0.29 and 0.22 with the final model, respectively, for the MIRAS and MR phases and Phaser Z scores of 4.0 and 4.3 for the rotation and translation searches), phase combination followed by density modification gave a readily interpretable map. MR with a distantly related model located a dimer in the asymmetric unit and provided the correct transformation to use in averaging electron density between SgrAI subunits. MIRAS data sets with low substitution and MR solutions from only distantly related models should not be ignored, as poor-quality starting phases can be significantly improved. The bootstrapping strategy employed to improve the initial MIRAS phases is described.
• Dunten, P. W., Little, E. J., Gregory, M. T., Manohar, V. M., Dalton, M., Hough, D., Bitinaite, J., & Horton, N. C. (2008). The structure of SgrAI bound to DNA; recognition of an 8 base pair target. Nucleic acids research, 36(16), 5405-16.
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  The three-dimensional X-ray crystal structure of the 'rare cutting' type II restriction endonuclease SgrAI bound to cognate DNA is presented. SgrAI forms a dimer bound to one duplex of DNA. Two Ca(2+) bind in the enzyme active site, with one ion at the interface between the protein and DNA, and the second bound distal from the DNA. These sites are differentially occupied by Mn(2+), with strong binding at the protein-DNA interface, but only partial occupancy of the distal site. The DNA remains uncleaved in the structures from crystals grown in the presence of either divalent cation. The structure of the dimer of SgrAI is similar to those of Cfr10I, Bse634I and NgoMIV, however no tetrameric structure of SgrAI is observed. DNA contacts to the central CCGG base pairs of the SgrAI canonical target sequence (CR|CCGGYG, | marks the site of cleavage) are found to be very similar to those in the NgoMIV/DNA structure (target sequence G|CCGGC). Specificity at the degenerate YR base pairs of the SgrAI sequence may occur via indirect readout using DNA distortion. Recognition of the outer GC base pairs occurs through a single contact to the G from an arginine side chain located in a region unique to SgrAI.
• Horton, N., Babic, A. C., Little, E. J., Manohar, V. M., Bitinaite, J., & Horton, N. C. (2008). DNA distortion and specificity in a sequence-specific endonuclease. Journal of molecular biology, 383(1).
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  Five new structures of the Q138F HincII enzyme bound to a total of three different DNA sequences and three different metal ions (Ca(2+), Mg(2+), and Mn(2+)) are presented. While previous structures were produced from soaking Ca(2+) into preformed Q138F HincII/DNA crystals, the new structures are derived from cocrystallization with Ca(2+), Mg(2+), or Mn(2+). The Mn(2)(+)-bound structure provides the first view of a product complex of Q138F HincII with cleaved DNA. Binding studies and a crystal structure show how Ca(2+) allows trapping of a Q138F HincII complex with noncognate DNA in a catalytically incompetent conformation. Many Q138F HincII/DNA structures show asymmetry, despite the binding of a symmetric substrate by a symmetric enzyme. The various complexes are fit into a model describing the different conformations of the DNA-bound enzyme and show how DNA conformational energetics determine DNA-cleavage rates by the Q138F HincII enzyme.
• Horton, N., Little, E. J., Babic, A. C., & Horton, N. C. (2008). Early interrogation and recognition of DNA sequence by indirect readout. Structure (London, England : 1993), 16(12).
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  Control of replication, transcription, recombination and repair requires proteins capable of finding particular DNA sequences in a background of a large excess of nonspecific sequences. Such recognition can involve direct readout, with direct contacts to the bases of DNA, or in some cases through the less well-characterized indirect readout mechanisms. In order to measure the relative contributions of direct and indirect readout by a sequence specific endonuclease, HincII, a mutant enzyme deficient in a direct contact, was characterized, and surprisingly showed no loss of sequence specificity. The three dimensional crystal structure shows the loss of most of the direct readout contacts to the DNA, possibly capturing an early stage in target site recognition using predominately indirect readout to prescreen sites before full sequence interrogation.
• Horton, N., Joshi, H. K., Etzkorn, C., Chatwell, L., Bitinaite, J., & Horton, N. C. (2006). Alteration of sequence specificity of the type II restriction endonuclease HincII through an indirect readout mechanism. The Journal of biological chemistry, 281(33).
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  The functional and structural consequences of a mutation of the DNA intercalating residue of HincII, Q138F, are presented. Modeling has suggested that the DNA intercalation by Gln-138 results in DNA distortions potentially used by HincII in indirect readout of its cognate DNA, GTYRAC (Y = C or T, R = A or G) (Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47). Kinetic data presented here indicate that the mutation of glutamine 138 to phenylalanine (Q138F) results in a change in sequence specificity at the center two base pairs of the cognate recognition site. We show that the preference of HincII for cutting, but not binding, the three cognate sites differing in the center two base pairs has been altered by the mutation Q138F. Five new crystal structures are presented including Q138F HincII bound to GTTAAC and GTCGAC both with and without Ca2+ as well as the structure of wild type HincII bound to GTTAAC. The Q138F HincII/DNA structures show conformational changes in the protein, bound DNA, and at the protein-DNA interface, consistent with the formation of adaptive complexes. Analysis of these structures and the effect of Ca2+ binding on the protein-DNA interface illuminates the origin of the altered specificity by the mutation Q138F in the HincII enzyme.
• Segal, D. J., Crotty, J. W., Bhakta, M. S., Barbas, C. F., & Horton, N. C. (2006). Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA. Journal of molecular biology, 363(2), 405-21.
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  Cys2-His2 zinc fingers are one of the most common types of DNA-binding domains. Modifications to zinc-finger binding specificity have recently enabled custom DNA-binding proteins to be designed to a wide array of target sequences. We present here a 1.96 A structure of Aart, a designed six-zinc finger protein, bound to a consensus DNA target site. This is the first structure of a designed protein with six fingers, and was intended to provide insights into the unusual affinity and specificity characteristics of this protein. Most protein-DNA contacts were found to be consistent with expectations, while others were unanticipated or insufficient to explain specificity. Several were unexpectedly mediated by glycerol, water molecules or amino acid-base stacking interactions. These results challenge some conventional concepts of recognition, particularly the finding that triplets containing 5'A, C, or T are typically not specified by direct interaction with the amino acid in position 6 of the recognition helix.
• Horton, N., Crotty, J. W., Etzkorn, C., Barbas, C. F., Segal, D. J., & Horton, N. C. (2005). Crystallization and preliminary X-ray crystallographic analysis of Aart, a designed six-finger zinc-finger peptide, bound to DNA. Acta crystallographica. Section F, Structural biology and crystallization communications, 61(Pt 6).
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  Crystals of a designed six-finger zinc-finger protein, Aart, bound to a 22-base-pair duplex DNA containing a consensus binding site have been obtained. Crystals grew by hanging-drop vapor diffusion from solutions containing polyethylene glycol 4000 as the precipitating agent. The irregularly shaped crystals belong to space group P1, with unit-cell parameters a = 41.95, b = 71.76, c = 74.73 A, alpha = 100.87, beta = 96.22, gamma = 106.33 degrees . There are most likely to be two protein-DNA complexes in the asymmetric unit. A complete native data set has been collected from a high-energy synchrotron source to a resolution of 2.95 A at 100 K, with an Rmerge of 9.3%.
• Horton, N., Little, E. J., & Horton, N. C. (2005). DNA-induced conformational changes in type II restriction endonucleases: the structure of unliganded HincII. Journal of molecular biology, 351(1).
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  The 2.1A crystal structure of the unliganded type II restriction endonuclease, HincII, is described. Although the asymmetric unit contains only a single monomer, crystal lattice contacts bring two monomers together to form a dimer very similar to that found in the DNA bound form. Comparison with the published DNA bound structure reveals that the DNA binding pocket is expanded in the unliganded structure. Comparison of the unliganded and DNA liganded structures reveals a simple rotation of subunits by 11 degrees each, or 22 degrees total, to a more closed structure around the bound DNA. Comparison of this conformational change to that observed in the other type II restriction endonucleases where DNA bound and unliganded forms have both been characterized, shows considerable variation in the types of conformational changes that can occur. The conformational changes in three can be described by a simple rotation of subunits, while in two others both rotation and translation of subunits relative to one another occurs. In addition, the endonucleases having subunits that undergo the greatest amount of rotation upon DNA binding are found to be those that distort the bound DNA the least, suggesting that DNA bending may be less facile in dimers possessing greater flexibility.
• Horton, N. C., & Perona, J. J. (2004). DNA cleavage by EcoRV endonuclease: two metal ions in three metal ion binding sites. Biochemistry, 43(22), 6841-57.
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  Four crystal structures of EcoRV endonuclease mutants K92A and K38A provide new insight into the mechanism of DNA bending and the structural basis for metal-dependent phosphodiester bond cleavage. The removal of a key active site positive charge in the uncleaved K92A-DNA-M(2+) substrate complex results in binding of a sodium ion in the position of the amine nitrogen, suggesting a key role for a positive charge at this position in stabilizing the sharp DNA bend prior to cleavage. By contrast, two structures of K38A cocrystallized with DNA and Mn(2+) ions in different lattice environments reveal cleaved product complexes featuring a common, novel conformation of the scissile phosphate group as compared to all previous EcoRV structures. In these structures, the released 5'-phosphate and 3'-OH groups remain in close juxtaposition with each other and with two Mn(2+) ions that bridge the conserved active site carboxylates. The scissile phosphates are found midway between their positions in the prereactive substrate and postreactive product complexes of the wild-type enzyme. Mn(2+) ions occupy two of the three sites previously described in the prereactive complexes and are plausibly positioned to generate the nucleophilic hydroxide ion, to compensate for the incipient additional negative charge in the transition state, and to ionize a second water for protonation of the 3'-oxyanion. Reconciliation of these findings with earlier X-ray and fluorescence studies suggests a novel mechanism in which a single initially bound metal ion in a third distinct site undergoes a shift in position together with movement of the scissile phosphate deeper into the active site cleft. This reconfigures the local environment to permit binding of the second metal ion followed by movement toward the pentacovalent transition state. The new mechanism suggested here embodies key features of previously proposed two- and three-metal catalytic models, and offers a view of the stereochemical pathway that integrates much of the copious structural and functional data that are available from exhaustive studies in many laboratories.
• Horton, N., & Perona, J. (2004). Crystallographic snapshots along a protein-induced DNA-bending pathway. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 97(11), 5729-5734.
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  Two new high-resolution cocrystal structures of EcoRV endonuclease bound to DNA show that a large variation in DNA-bending angles is sampled in the ground state binary complex. Together with previous structures, these data reveal a contiguous series of protein conformational states delineating a specific trajectory for the induced-fit pathway. Rotation of the DNA-binding domains, together with movements of two symmetry-related helices binding in the minor groove, causes base unstacking at a key base-pair step and propagates structural changes that assemble the active sites. These structures suggest a complex mechanism for DNA bending that depends on forces generated by interacting protein segments, and on selective neutralization of phosphate charges along the inner face of the bent double helix.
• Horton, N., Etzkorn, C., & Horton, N. C. (2004). Ca2+ binding in the active site of HincII: implications for the catalytic mechanism. Biochemistry, 43(42).
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  The 2.8 A crystal structure of the type II restriction endonuclease HincII bound to Ca(2+) and cognate DNA containing GTCGAC is presented. The DNA is uncleaved, and one calcium ion is bound per active site, in a position previously described as site I in the related blunt cutting type II restriction endonuclease EcoRV [Horton, N. C., Newberry, K. J., and Perona, J. J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95 (23), 13489-13494], as well as that found in other related enzymes. Unlike the site I metal in EcoRV, but similar to that of PvuII, NgoMIV, BamHI, BglII, and BglI, the observed calcium cation is directly ligated to the pro-S(p) oxygen of the scissile phosphate. A calcium ion-ligated water molecule is well positioned to act as the nucleophile in the phosphodiester bond cleavage reaction, and is within hydrogen bonding distance of the conserved active site lysine (Lys 129), as well as the pro-R(p) oxygen of the phosphate group 3' of the scissile phosphate, suggesting possible roles for these groups in the catalytic mechanism. Kinetic data consistent with an important role for the 3'-phosphate group in DNA cleavage by HincII are presented. The previously observed sodium ion [Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47] persists in the active sites of the Ca(2+)-bound structure; however, kinetic data show little effect on the single-turnover rate of DNA cleavage in the absence of Na(+) ions.
• Horton, N., Etzkorn, C., & Horton, N. C. (2004). Mechanistic insights from the structures of HincII bound to cognate DNA cleaved from addition of Mg2+ and Mn2+. Journal of molecular biology, 343(4).
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  The three-dimensional X-ray crystal structures of HincII bound to cognate DNA containing GTCGAC and Mn(2+) or Mg(2+), at 2.50A and 2.95A resolution, respectively, are presented. In both structures, the DNA is found cleaved, and the positions of the active-site groups, cleaved phosphate group, and 3' oxygen atom of the leaving group are in very similar positions. Two highly occupied Mn(2+) positions are found in each active site of the four crystallographically independent subunit copies in the HincII/DNA/Mn(2+) structure. The manganese ion closest to the previously identified single Ca(2+) position of HincII is shifted 1.7A and has lost direct ligation to the active-site aspartate residue, Asp127. A Mn(2+)-ligated water molecule in a position analogous to that seen in the HincII/DNA/Ca(2+) structure, and proposed to be the attacking nucleophile, is beyond hydrogen bonding distance from the active-site lysine residue, Lys129, but remains within hydrogen bonding distance from the proRp oxygen atom of the phosphate group 3' to the scissile phosphate group. In addition, the position of the cleaved phosphate group is on the opposite side of the axis connecting the two metal ions relative to that found in the BamHI/product DNA/Mn(2+) structure. Mechanistic implications are discussed, and a model for the two-metal-ion mechanism of DNA cleavage by HincII is proposed.
• Horton, N. C., Dorner, L. F., & Perona, J. J. (2002). Sequence selectivity and degeneracy of a restriction endonuclease mediated by DNA intercalation. Nature structural biology, 9(1), 42-7.
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  The crystal structure of the HincII restriction endonuclease-DNA complex shows that degenerate specificity for blunt-ended cleavage at GTPyPuAC sequences arises from indirect readout of conformational preferences at the center pyrimidine-purine step. Protein-induced distortion of the DNA is accomplished by intercalation of glutamine side chains into the major groove on either side of the recognition site, generating bending by either tilt or roll at three distinct loci. The intercalated side chains propagate a concerted shift of all six target-site base pairs toward the minor groove, producing an unusual cross-strand purine stacking at the center pyrimidine-purine step. Comparison of the HincII and EcoRV cocrystal structures suggests that sequence-dependent differences in base-stacking free energies are a crucial underlying factor mediating protein recognition by indirect readout.
• Horton, N. C., Otey, C., Lusetti, S., Sam, M. D., Kohn, J., Martin, A. M., Ananthnarayan, V., & Perona, J. J. (2002). Electrostatic contributions to site specific DNA cleavage by EcoRV endonuclease. Biochemistry, 41(35), 10754-63.
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  Mutational analysis of amino acids at the periphery of the EcoRV endonuclease active site suggests that moderate-range electrostatic effects play a significant role in modulating the efficiency of phosphoryl transfer. Asp36 and Lys38 located on minor-groove binding surface loops approach within 7-9 A of the scissile phosphates of the DNA. While the rates of single-site mutations removing the carboxylate or amine moieties at these positions are decreased 10(3)-10(5)-fold compared to that of wild-type EcoRV, we find that double mutants which rebalance the charge improve catalysis by up to 500-fold. Mutational analysis also suggests that catalytic efficiency is influenced by Lys173, which is buried at the base of a deep depression penetrating from a distal surface of the enzyme. The Lys173 amine group lies just 6 A from the amine group of the conserved essential Lys92 side chain in the active site. Kinetic and crystallographic analyses of the EcoRV E45A mutant enzyme further show that the Glu45 carboxylate group facilitates an extensive set of conformational transitions which occur upon DNA binding. The crystal structure of E45A bound to DNA and Mn2+ ions reveals significant conformational alterations in a small alpha-helical portion of the dimer interface located adjacent to the DNA minor groove. This leads to a tertiary reorientation of the two monomers as well as shifting of the key major-groove binding recognition loops. Because the Glu45 side chain does not appear to play a direct structural role in maintaining the active site, these rearrangements may instead originate in an altered electrostatic potential caused by removal of the negative charge. A Mn2+ binding site on the scissile phosphate is also disrupted in the E45A structure such that inner-sphere metal interactions made by the scissile DNA phosphate and conserved Asp90 carboxylate are each replaced with water molecules in the mutant. These findings argue against a proposed role for Asp36 as the general base in EcoRV catalysis, and reveal that the induced-fit conformational changes necessary for active site assembly and metal binding are significantly modulated by the electrostatic potential in this region.
• Horton, N., & Perona, J. (2002). Making the most of metal ions. NATURE STRUCTURAL BIOLOGY, 8(4), 290-293.
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  Crystal structures of the homing endonuclease I-Crel bound to substrate DNA and divalent metals show that one metal ion is shared between the two active sites of the enzyme. This arrangement appears uniquely suited to the formation of double-stranded DNA breaks via a concerted reaction.
• Sam, M., Horton, N., Nissan, T., & Perona, J. (2002). Catalytic efficiency and sequence selectivity of a restriction endonuclease modulated by a distal manganese ion binding site. JOURNAL OF MOLECULAR BIOLOGY, 306(4), 851-861.
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  Crystal structures of EcoRV endonuclease bound in a ternary complex with cognate duplex DNA and manganese ions have previously revealed an Mn2+-binding site located between the enzyme and the DNA outside of the dyad-symmetric GATATC recognition sequence. In each of the two enzyme subunits, this metal ion bridges between a distal phosphate group of the DNA and the imidazole ring of His71. The new metal-binding site is specific to Mn2+ and is not occupied in ternary cocrystal structures with either Mg2+ or Ca2+. Characterization of the H71A and H71Q mutants of EcoRV now demonstrates that these distal Mn2+ sites significantly modulate activity toward both cognate and non-cognate DNA substrates. Single-turnover and steady-state kinetic analyses show that removal of the distal site in the mutant enzymes increases Mn2+ dependent cleavage rates of specific substrates by tenfold. Conversely, the enhancement of non-cognate cleavage at GTTATC sequences by Mn2+ is significantly attenuated in the mutants. As a consequence, under Mn2+ conditions EcoRV-H71A and EcoRV-H71Q are 100 to 700-fold more specific than the wild-type enzyme for cognate DNA relative to the GTTATC non-cognate site. These data reveal a strong dependence of DNA cleavage efficiency upon metal ion-mediated interactions located some 20 Angstrom distant from the scissile phosphodiester linkages. They also show that discrimination of cognate versus non-cognate DNA sequences by EcoRV depends in part on contacts with the sugar-phosphate backbone outside of the target site. (C) 2002 Academic Press.
• Horton, N. C., & Perona, J. J. (2001). Making the most of metal ions. Nature structural biology, 8(4), 290-3.
• Horton, N., Connolly, B., & Perona, J. (2001). Inhibition of EcoRV endonuclease by deoxyribo-3 '-S-phosphorothiolates: A high-resolution X-ray crystallographic study. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 122(14), 3314-3324.
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  Three high-resolution structures of the restriction endonuclease EcoRV bound to a duplex DNA substrate analogue with deoxyribo-3'-S-phosphorothiolate linkages at both scissile phosphates are presented. In each of these structures cocrystallized with Mg2+, Mn2+, or Ca2+ ions, the nonesterified pro-S oxygen of the scissile phosphate no longer directly ligates a divalent cation, as is observed for the unmodified complex. Instead, one metal ion in all three structures is shifted toward the adjacent 3'-phosphate of the DNA, to occupy a position nearly identical to that previously observed in an EroRV T93A/DNA/Ca2+ complex (N. C. Horton et al.. Proc. Natl. Acad Sci. U.S.A. 1998, 95, 13489). A second divalent metal ion in each structure bridges the carboxylate groups of Asp74 and Glu45 (74/45 site), as also seen in both wild-type and T93A cocrystals. The uncleaved 3'-S-phosphorothiolate DNAs in these complexes are only slightly distorted from the conformation of the unmodified duplex. Kinetic measurements show that the rate of the chemical step For analogue cleavage is severely reduced for each of the active metals Mg2+, Mn2+, and Co2+, and that the thiophilic Mn2+, Cd2+, and Zn2+ cations do not provide a measurable reconstitution of activity. The inability of thiophilic metals to improve activity is consistent with models for catalysis derived from previous crystal structures, which indicate that ligation of a metal ion to the 3'-oxygen is mediated through an inner-sphere water molecule rather than by direct interaction. The structures suggest that 3'-S-phosphorothiolale analogues resist cleavage because the bridging sulfur excludes inner-sphere ligation of divalent metal ions to any position on the scissile phosphate. This distinguishes the inhibitory mechanism in EcoRV from that operative in the 3'-5' exonuclease active site of DNA polymerase I (C. A. Brautigam et al., Biochemistry, 1999, 38, 696), and likely as well from other enzymes which also catalyze phosphoryl transfer via direct metal ligation to the 3'-oxygen leaving group.
• Horton, N., Dorner, L., & Perona, J. (2001). Sequence selectivity and degeneracy of a restriction endonuclease mediated by DNA intercalation. NATURE STRUCTURAL BIOLOGY, 9(1), 42-47.
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  The crystal structure of the HincII restriction endonuclease- DNA complex shows that degenerate specificity for blunt-ended cleavage at GTPyPuAC sequences arises from indirect readout of conformational preferences at the center pyrimidine-purine step. Protein-induced distortion of the DNA is accomplished by intercalation of glutamine side chains into the major groove on either side of the recognition site, generating bending by either tilt or roll at three distinct loci. The intercalated side chains propagate a concerted shift of all six target-site base pairs toward the minor groove, producing an unusual cross-strand purine stacking at the center pyrimidine-purine step. Comparison of the HincII and EcoRV cocrystal structures suggests that sequence-dependent differences in base-stacking free energies are a crucial underlying factor mediating protein recognition by indirect readout.
• Sam, M. D., Horton, N. C., Nissan, T. A., & Perona, J. J. (2001). Catalytic efficiency and sequence selectivity of a restriction endonuclease modulated by a distal manganese ion binding site. Journal of molecular biology, 306(4), 851-61.
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  Crystal structures of EcoRV endonuclease bound in a ternary complex with cognate duplex DNA and manganese ions have previously revealed an Mn(2+)-binding site located between the enzyme and the DNA outside of the dyad-symmetric GATATC recognition sequence. In each of the two enzyme subunits, this metal ion bridges between a distal phosphate group of the DNA and the imidazole ring of His71. The new metal- binding site is specific to Mn(2+) and is not occupied in ternary cocrystal structures with either Mg(2+) or Ca(2+). Characterization of the H71A and H71Q mutants of EcoRV now demonstrates that these distal Mn(2+) sites significantly modulate activity toward both cognate and non-cognate DNA substrates. Single-turnover and steady-state kinetic analyses show that removal of the distal site in the mutant enzymes increases Mn(2+)-dependent cleavage rates of specific substrates by tenfold. Conversely, the enhancement of non-cognate cleavage at GTTATC sequences by Mn(2+) is significantly attenuated in the mutants. As a consequence, under Mn(2+) conditions EcoRV-H71A and EcoRV-H71Q are 100 to 700-fold more specific than the wild-type enzyme for cognate DNA relative to the GTTATC non-cognate site. These data reveal a strong dependence of DNA cleavage efficiency upon metal ion-mediated interactions located some 20 A distant from the scissile phosphodiester linkages. They also show that discrimination of cognate versus non-cognate DNA sequences by EcoRV depends in part on contacts with the sugar-phosphate backbone outside of the target site.
• Horton, N. C., & Perona, J. J. (2000). Crystallographic snapshots along a protein-induced DNA-bending pathway. Proceedings of the National Academy of Sciences of the United States of America, 97(11), 5729-34.
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  Two new high-resolution cocrystal structures of EcoRV endonuclease bound to DNA show that a large variation in DNA-bending angles is sampled in the ground state binary complex. Together with previous structures, these data reveal a contiguous series of protein conformational states delineating a specific trajectory for the induced-fit pathway. Rotation of the DNA-binding domains, together with movements of two symmetry-related helices binding in the minor groove, causes base unstacking at a key base-pair step and propagates structural changes that assemble the active sites. These structures suggest a complex mechanism for DNA bending that depends on forces generated by interacting protein segments, and on selective neutralization of phosphate charges along the inner face of the bent double helix.
• Horton, N., & Perona, J. (2000). Recognition of flanking DNA sequences by EcoRV endonuclease involves alternative patterns of water-mediated contacts. JOURNAL OF BIOLOGICAL CHEMISTRY, 273(34), 21721-21729.
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  The 2.1-Angstrom cocrystal structure of EcoRV endonuclease bound to 5'-CGGGATATCCC, in a crystal lattice isomorphous with the cocrystallized undecamer 5'-AAAGATATCTT previously determined, shows novel base recognition in the major groove of the DNA flanking the GATATC target site. Lys(104) Of the enzyme interacts through water molecules with the exocyclic N-4 amino groups of flanking cytosines. Steric exclusion of water molecule-binding sites by the 5-methyl group of thymine drives the adoption of alternative water-mediated contacts with AT versus GC flanks. This structure provides a rare example of structural adaptability in the recognition of different DNA sequences by a protein and suggests preferred strategies for the expansion of target site specificity by EcoRV.
• Horton, N., & Perona, J. (2000). Role of protein-induced bending in the specificity of DNA recognition: Crystal structure of EcoRV endonuclease complexed with d(AAAGAT)+d(ATCTT). JOURNAL OF MOLECULAR BIOLOGY, 277(4), 779-787.
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  The crystal structure of EcoRV endonuclease has been determined at 2.1 Angstrom resolution complexed to two five-base-pair DNA duplexes each containing the cognate recognition half-site. The highly localized 50 degrees bend into the major groove seen at the center TA-step of the continuous GATATC site is preserved in this discontinuous DNA complex lacking the scissile phosphates. Thus, this crystal structure provides evidence that covalent constraints associated with a continuous target site are not essential to enzyme-induced DNA bending, even when these constraints are removed directly at the locus of the bend. The scissile phosphates are also absent in the crystal structure of EcoRV bound to the non-specific site TCGCGA, which shows a straight B-like conformation. We conclude that DNA bending by EcoRV is governed only by the sequence and is not influenced by the continuity of the phosphodiester backbone. Together with other data showing that cleavable non-cognate sites are bent, these results indicate that EcoRV bends non-cognate sites differing by one or two base-pairs from GATATC, but does not bend non-specific sites that are less similar. Structural and thermodynamic considerations suggest that the sequence-dependent energy cost of DNA bending is likely to Flay an important role in determining the specificity of EcoRV. This differential cost is manifested at the binding step for bent non-cognate sequences and at the catalytic step for unbent non-specific sequences. (C) 1998 Academic Press Limited.
• Horton, N., Dorner, L., Schildkraut, ., & Perona, J. (2000). Crystallization and preliminary diffraction analysis of the HincII restriction endonuclease-DNA complex. ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 55, 1943-1945.
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  Crystals of the 60 kDa dimeric HincII restriction enzyme bound to a 12 base-pair dyad-symmetric duplex DNA carrying the specific 5'-GTCGAC recognition site have been obtained. Crystals grew by hanging-drop vapor diffusion from solutions containing polyethylene glycol 4000 as precipitating agent. The rod-shaped crystals belong to space group I222 (or I2(1)2(1)2(1)), With unit-cell dimensions a = 66.9, b = 176.7, c = 256.0 Angstrom. There are most likely to be two dimeric complexes in the asymmetric unit. A complete native data set has been collected from a high-energy synchrotron source to a resolution of 2.5 Angstrom at 100 K, with an R-merge of 4.8%.
• Horton, N. C., Dorner, L. F., Schildkraut, I., & Perona, J. J. (1999). Crystallization and preliminary diffraction analysis of the HincII restriction endonuclease-DNA complex. Acta crystallographica. Section D, Biological crystallography, 55(Pt 11), 1943-5.
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  Crystals of the 60 kDa dimeric HincII restriction enzyme bound to a 12 base-pair dyad-symmetric duplex DNA carrying the specific 5'-GTCGAC recognition site have been obtained. Crystals grew by hanging-drop vapor diffusion from solutions containing polyethylene glycol 4000 as precipitating agent. The rod-shaped crystals belong to space group I222 (or I2(1)2(1)2(1)), with unit-cell dimensions a = 66.9, b = 176.7, c = 256.0 A. There are most likely to be two dimeric complexes in the asymmetric unit. A complete native data set has been collected from a high-energy synchrotron source to a resolution of 2.5 A at 100 K, with an R(merge) of 4.8%.
• Horton, N., & Perona, J. (1999). DNA cleavage by EcoRV endonuclease: Two metal ions in three metal ion binding sites. BIOCHEMISTRY, 43(22), 6841-6857.
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  Four crystal structures of EcoRV endonuclease mutants K92A and K38A provide new insight into the mechanism of DNA bending and the structural basis for metal-dependent phosphodiester bond cleavage. The removal of a key active site positive charge in the uncleaved K92A-DNA-M2+ substrate complex results in binding of a sodium ion in the position of the amine nitrogen, suggesting a key role for a positive charge at this position in stabilizing the sharp DNA bend prior to cleavage. By contrast, two structures of K38A cocrystallized with DNA and Mn2+ ions in different lattice environments reveal cleaved product complexes featuring a common, novel conformation of the scissile phosphate group as compared to all previous EcoRV structures. In these structures, the released 5'-phosphate and 3'-OH groups remain in close juxtaposition with each other and with two Mn2+ ions that bridge the conserved active site carboxylates. The scissile phosphates are found midway between their positions in the prereactive substrate and postreactive product complexes of the wild-type enzyme. Mn2+ ions occupy two of the three sites previously described in the prereactive complexes and are plausibly positioned to generate the nucleophilic hydroxide ion, to compensate for the incipient additional negative charge in the transition state, and to ionize a second water for protonation of the 3'-oxyanion. Reconciliation of these findings with earlier X-ray and fluorescence studies suggests a novel mechanism in which a single initially bound metal ion in a third distinct site undergoes a shift in position together with movement of the scissile phosphate deeper into the active site cleft. This reconfigures the local environment to permit binding of the second metal ion followed by movement toward the pentacovalent transition state. The new mechanism suggested here embodies key features of previously proposed two- and three-metal catalytic models, and offers a view of the stereochemical pathway that integrates much of the copious structural and functional data that are available from exhaustive studies in many laboratories.
• Martin, A. M., Horton, N. C., Lusetti, S., Reich, N. O., & Perona, J. J. (1999). Divalent metal dependence of site-specific DNA binding by EcoRV endonuclease. Biochemistry, 38(26), 8430-9.
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  Measurements of binding equilibria of EcoRV endonuclease to DNA, for a series of base-analogue substrates, demonstrate that expression of sequence selectivity is strongly enhanced by the presence of Ca2+ ions. Binding constants were determined for short duplex oligodeoxynucleotides containing the cognate DNA site, three cleavable noncognate sites, and a fully nonspecific site. At pH 7.5 and 100 mM NaCl, the full range of specificity from the specific (tightest binding) to nonspecific (weakest binding) sites is 0.9 kcal/mol in the absence of metal ions and 5.8 kcal/mol in the presence of Ca2+. Precise determination of binding affinities in the presence of the active Mg2+ cofactor was found to be possible for substrates retaining up to 1.6% of wild-type activity, as determined by the rate of phosphoryl transfer. These measurements show that Ca2+ is a near-perfect analogue for Mg2+ in binding reactions of the wild-type enzyme with DNA base-analogue substrates, as it provides identical DeltaDeltaG degrees bind values among the cleavable noncognate sites. Equilibrium dissociation constants of wild-type and base-analogue sites were also measured for the weakly active EcoRV mutant K38A, in the presence of either Mg2+ or Ca2+. In this case, Ca2+ allows expression of a greater degree of specificity than does Mg2+. DeltaDeltaG degrees bind values of K38A toward specific versus nonspecific sites are 6.1 kcal/mol with Ca2+ and 3.9 kcal/mol with Mg2+, perhaps reflecting metal-specific conformational changes in the ground-state ternary complexes. The enhancement of binding specificity provided by divalent metal ions is likely to be general to many restriction endonucleases and other metal-dependent nucleic acid-modifying enzymes. These results strongly suggest that measurements of DNA binding affinities for EcoRV, and likely for many other restriction endonucleases, should be performed in the presence of divalent metal ions.
• Baldwin, E. T., Sarver, R. W., Bryant, G. L., Curry, K. A., Fairbanks, M. B., Finzel, B. C., Garlick, R. L., Heinrikson, R. L., Horton, N. C., Kelley, L. L., Mildner, A. M., Moon, J. B., Mott, J. E., Mutchler, V. T., Tomich, C. S., Watenpaugh, K. D., & Wiley, V. H. (1998). Cation binding to the integrin CD11b I domain and activation model assessment. Structure (London, England : 1993), 6(7), 923-35.
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  The integrin family of cell-surface receptors mediate cell adhesion through interactions with the extracellular matrix or other cell-surface receptors. The alpha chain of some integrin heterodimers includes an inserted 'I domain' of about 200 amino acids which binds divalent metal ions and is essential for integrin function. Lee et al. proposed that the I domain of the integrin CD11b adopts a unique 'active' conformation when bound to its counter receptor. In addition, they proposed that the lack of adhesion in the presence of Ca2+ ion reflected the stabilization of an 'inactive' I-domain conformation. We set out to independently determine the structure of the CD11 b I domain and to evaluate the structural effects of divalent ion binding to this protein.
• Horton, N. C., & Perona, J. J. (1998). Recognition of flanking DNA sequences by EcoRV endonuclease involves alternative patterns of water-mediated contacts. The Journal of biological chemistry, 273(34), 21721-9.
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  The 2.1-A cocrystal structure of EcoRV endonuclease bound to 5'-CGGGATATCCC, in a crystal lattice isomorphous with the cocrystallized undecamer 5'-AAAGATATCTT previously determined, shows novel base recognition in the major groove of the DNA flanking the GATATC target site. Lys104 of the enzyme interacts through water molecules with the exocyclic N-4 amino groups of flanking cytosines. Steric exclusion of water molecule-binding sites by the 5-methyl group of thymine drives the adoption of alternative water-mediated contacts with AT versus GC flanks. This structure provides a rare example of structural adaptability in the recognition of different DNA sequences by a protein and suggests preferred strategies for the expansion of target site specificity by EcoRV.
• Horton, N. C., & Perona, J. J. (1998). Role of protein-induced bending in the specificity of DNA recognition: crystal structure of EcoRV endonuclease complexed with d(AAAGAT) + d(ATCTT). Journal of molecular biology, 277(4), 779-87.
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  The crystal structure of EcoRV endonuclease has been determined at 2. 1 A resolution complexed to two five-base-pair DNA duplexes each containing the cognate recognition half-site. The highly localized 50 degrees bend into the major groove seen at the center TA-step of the continuous GATATC site is preserved in this discontinuous DNA complex lacking the scissile phosphates. Thus, this crystal structure provides evidence that covalent constraints associated with a continuous target site are not essential to enzyme-induced DNA bending, even when these constraints are removed directly at the locus of the bend. The scissile phosphates are also absent in the crystal structure of EcoRV bound to the non-specific site TCGCGA, which shows a straight B-like conformation. We conclude that DNA bending by EcoRV is governed only by the sequence and is not influenced by the continuity of the phosphodiester backbone. Together with other data showing that cleavable non-cognate sites are bent, these results indicate that EcoRV bends non-cognate sites differing by one or two base-pairs from GATATC, but does not bend non-specific sites that are less similar. Structural and thermodynamic considerations suggest that the sequence-dependent energy cost of DNA bending is likely to play an important role in determining the specificity of EcoRV. This differential cost is manifested at the binding step for bent non-cognate sequences and at the catalytic step for unbent non-specific sequences.
• Horton, N. C., Newberry, K. J., & Perona, J. J. (1998). Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases. Proceedings of the National Academy of Sciences of the United States of America, 95(23), 13489-94.
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  The 2.15-A resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3' to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-SP phosphoryl oxygen on the adjacent 3' phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.
• Horton, N., Newberry, K., & Perona, J. (1998). Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 95(23), 13489-13494.
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  The 2.15-Angstrom resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3' to the scissile phosphate. A second inner-sphere mater on this metal is positioned approximately in-line for attack on the scissile phosphate, This structure corroborates the observation that the pro-S-P phosphoryl oxygen on the adjacent 3' phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step hp a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.
• Horton, N., Otey, C., Lusetti, S., Sam, M., Kohn, J., Martin, A., Ananthnarayan, ., & Perona, J. (1998). Electrostatic contributions to site specific DNA cleavage by EcoRV endonuclease. BIOCHEMISTRY, 41(35), 10754-10763.
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  Mutational analysis of amino acids at the periphery of the EcoRV endonuclease active site suggests that moderate-range electrostatic effects play a significant role in modulating the efficiency of phosphoryl transfer. Asp36 and Lys38 located on minor-groove binding surface loops approach within 7-9 Angstrom of the scissile phosphates of the DNA. While the rates of single-site mutations removing the carboxylate or amine moieties at these positions are decreased 10(3)-10(5)-fold compared to that of wildtype EcoRV, we find that double mutants which rebalance the charge improve catalysis by up to 500-fold. Mutational analysis also suggests that catalytic efficiency is influenced by Lys173, which is buried at the base of a deep depression penetrating from a distal surface of the enzyme. The Lys173 amine group lies just 6 A from the amine group of the conserved essential Lys92 side chain in the active site. Kinetic and crystallographic analyses of the EcoRV E45A mutant enzyme further show that the Glu45 carboxylate group facilitates an extensive set of conformational transitions which occur upon DNA binding. The crystal structure of E45A bound to DNA and Mn2+ ions reveals significant conformational alterations in a small alpha-helical portion of the dimer interface located adjacent to the DNA minor groove. This leads to a tertiary reorientation of the two monomers as well as shifting of the key major-groove binding recognition loops. Because the Glu45 side chain does not appear to play a direct structural role in maintaining the active site, these rearrangements may instead originate in an altered electrostatic potential caused by removal of the negative charge. A Mn2+ binding site on the scissile phosphate is also disrupted in the E45A structure such that inner-sphere metal interactions made by the scissile DNA phosphate and conserved Asp90 carboxylate are each replaced with water molecules in the mutant. These findings argue against a proposed role for Asp36 as the general base in EcoRV catalysis, and reveal that the induced-fit conformational changes necessary for active site assembly and metal binding are significantly modulated by the electrostatic potential in this region.
• Martin, A., Horton, N., Lusetti, S., Reich, N., & Perona, J. (1998). Divalent metal dependence of site-specific DNA binding by EcoRV endonuclease. BIOCHEMISTRY, 38(26), 8430-8439.
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  Measurements of binding equilibria of EcoRV endonuclease to DNA, for a series of base analogue substrates, demonstrate that expression of sequence selectivity is strongly enhanced by the presence of Ca2+ ions. Binding constants were determined for short duplex oligodeoxynucleotides containing the cognate DNA site, three cleavable noncognate sites, and a fully nonspecific site. At pH 7.5 and 180 mM NaCl, the full range of specificity from the specific (tightest binding) to nonspecific (weakest binding) sites is 0.9 kcal/mol in the absence of metal ions and 5.8 kcal/mol in the presence of Ca2+. Precise determination of binding affinities in the presence of the active Mg2+ cofactor was found to be possible for substrates retaining up to 1.6% of wild-type activity, as determined by the rare of phosphoryl transfer. These measurements show that Ca2+ is a near-perfect analogue for Mg2+ in binding reactions of the wild-type enzyme with DNA base-analogue substrates, as it provides identical Delta Delta G degrees(bind) values among the cleavable noncognate sites. Equilibrium dissociation constants of wild-type and base-analogue sites were also measured for the weakly active EcoRV mutant K38A, in the presence of either Mg2+ or Ca2+ In this case, Ca2+ allows expression of a greater degree of specificity than does Mg2+. Delta Delta G degrees(bind) values of K38A toward specific versus nonspecific sites are 6.1 kcal/mol with Ca2+ and 3.9 kcal/mol with Mg2+ perhaps reflecting metal-specific conformational changes in the ground-state ternary complexes. The enhancement of binding specificity provided by divalent metal ions is likely to be general to many restriction endonucleases and other metal-dependent nucleic acid-modifying enzymes. These results strongly suggest that measurements of DNA binding affinities for EcoRV, and likely for many other restriction endonucleases, should be performed in the presence of divalent metal ions.
• Horton, N., Lewis, M., & Lu, P. (1997). Escherichia coli lac repressor-lac operator interaction and the influence of allosteric effectors. JOURNAL OF MOLECULAR BIOLOGY, 265(1), 1-7.
• Horton, N., Lewis, M., & Lu, P. (1997). Escherichia coli lac repressor-lac operator interaction and the influence of allosteric effectors. Journal of molecular biology, 265(1), 1-7.
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  The wild type E. coli lac operator is embedded in a 35 base-pair DNA sequence containing extensive 2-fold symmetry, suggesting a symmetric repressor operator complex. However, deviations from strict 2-fold symmetry occur at the central base-pair and at three additional base-pairs. Using an operator fragment binding analysis we have determined: (a) a relative contribution each pair provides to the lac repressor-lac operator DNA complex, (b) the operator DNA length necessary for maximum binding to lac repressor; and (c) the contribution of the several non-symmetric base in the wild-type operator to the binding affinity. Since lac repressor-lac operator DNA interaction is reduced upon binding of the gratuitous inducer, isopropyl-beta-D-galactoside (IPTG), the same DNA fragment binding analysis was performed with the low affinity form of lac repressor. In the presence of inducer, the affinity for the left half site of the wild-type lac operator is reduced without significant reduction on the right half of the operator. Conversely, the anti-inducer orthonitrophenylfucoside (ONPF) which stabilizes the lac repressor-lac operator complex increases the binding affinity, particularly to the right half of the operator.
• Horton, N. C., & Finzel, B. C. (1996). The structure of an RNA/DNA hybrid: a substrate of the ribonuclease activity of HIV-1 reverse transcriptase. Journal of molecular biology, 264(3), 521-33.
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  The structure of a complementary hybrid duplex of RNA and DNA has been determined by X-ray crystallography. A ten residue DNA oligonucleotide of sequence 5'-G-G-C-G-C-C-C-G-A-A-3' was annealed to complementary RNA (5'-u-u-c-g-g-g-c-g-c-c-3') and crystallized, producing tetragonal crystals that diffract to 2.3 A resolution. The hybrid adopts a geometry that is neither strictly A nor B-form, rather the helix possesses qualities of both, reminiscent of spectroscopic descriptions of a hybrid conformation, or H-form. All of the ribonucleotides maintain the C3'-endo conformation seen in A-form, while both C3'-endo and C2'-endo conformations are found in the deoxyribonucleotides. The minor groove width (8.5 to 10.5 A) is intermediate between standard values for A (11 A) and B-form (7.4 A) DNA. The global parameters rise and base-pairs tilt (or inclination) are like that of A-DNA, however the slide and x displacement (Dx) are more like that of A-RNA, thus giving the hybrid a unique conformation. In addition, the 10-mer crystallizes in a manner that allows the formation of dimers that stack end-to-end, thereby providing a glimpse of how an extended (20 base-pair) helix of RNA-DNA hybrid might appear. This duplex sequence was selected for study because it is specifically recognized by the ribonuclease H function of HIV reverse transcriptase. A structure of a substrate of this enzyme is of potential value in understanding requirements for the selectivity of this important drug target. The minor groove of the hybrid duplex, lined with the 2-OH of the ribose rings, is the single distinguishing characteristic of the RNA/DNA hybrid, undoubtedly an important structural feature conferring selectivity.
• Lewis, M., Chang, G., Horton, N. C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., & Lu, P. (1996). Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science (New York, N.Y.), 271(5253), 1247-54.
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  The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-beta-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.
• HORTON, N., & LEWIS, M. (1992). CALCULATION OF THE FREE-ENERGY OF ASSOCIATION FOR PROTEIN COMPLEXES. PROTEIN SCIENCE, 1(1), 169-181.
• Horton, N., & Lewis, M. (1992). Calculation of the free energy of association for protein complexes. Protein science : a publication of the Protein Society, 1(1), 169-81.
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  We have developed a method for calculating the association energy of quaternary complexes starting from their atomic coordinates. The association energy is described as the sum of two solvation terms and an energy term to account for the loss of translational and rotational entropy. The calculated solvation energy, using atomic solvation parameters and the solvent accessible surface areas, has a correlation of 96% with experimentally determined values. We have applied this methodology to examine intermediates in viral assembly and to assess the contribution isomerization makes to the association energy of molecular complexes. In addition, we have shown that the calculated association can be used as a predictive tool for analyzing modeled molecular complexes.

Proceedings Publications

• Ghadirian, N., & Horton, N. C. (2020, April). Structure‐Function Studies of the Helicase Domain of NS1 Protein of Human Parvovirus B19. In Experimental Biology 2020 ASBMB, 34, 1.
• Horton, N. C. (2020, April). Filament Formation Induces a Shape Change and Activation of the Nuclease SgrAI. In Experimental Biology 2020 ASBMB, 34, 1.
• Horton, N. C. (2020, May). The Filament Forming Mechanism of SgrAI Endonuclease‐Structural and Kinetic Analysis. In Experimental Biology 2020 ASBMB, 34, 1.

Presentations

• Horton, N. C. (2022). I presented my research at 3 conferences in oral form:
  1. Enzymes, Coenzymes and Metabolic Pathways GRC
  2. The Protein Society
  3. Experimental Biology/ASBMB 2022
  I also presented posters at these meetings, and brought 2 students to the Experimental Biology meeting in 2022.. GRC and Protein Society and Experimental Biology.
• Horton, N. C. (2018, summer). DNA binding and cleavage by human parvovirus B19 NS1 nuclease domain. XVII International Parvovirus Workshop. Miami, Florida.
• Horton, N. C. (2019, April). Run-On Oligomerization, a Novel Strategy to Control Enzyme Activity and Specificity. Experimental Biology/ASBMB Joint Meeting. Orlando, FL: FASEB/ASBMB.
• Horton, N. C. (2019, March). Run-On Oligomerization, a Novel Strategy to Control Enzyme Activity and Specificity. West Coast Structural Biology Workshop. Asilomar, CA: WCSBW.
• Horton, N. C. (2019, September). Enzyme Filamentation: A New Enzyme Regulatory Mechanism and So Much More. MCB Seminar Series. Tucson, AZ: MCB Department.
• Horton, N. C. (2019, September). Run-On Oligomerization, a Novel Strategy to Control Enzyme Activity and Specificity. Toyota Physical and Chemical Research Institute Workshop. Nagakuti, Japan: Toyota Riken Research Institute.
• Horton, N. C. (2018, June). Run-On Oligomerization, a Novel Strategy to Control Enzyme Activity and Specificity. FASEB Machines on Genes. Aspen, CO: FASEB.
• Horton, N. C. (2018, May). Run-On Oligomerization, a Novel Strategy to Control Enzyme Activity and Specificity. MCB Seminar Series. Tucson, AZ: MCB Department.
• Horton, N. C. (2018, spring). Run-on oligomerization, a novel strategy to control enzyme activity and specificity. MCB Colloquium, University of Arizona. Tucson, AZ: MCB department.
• Horton, N. C. (2018, summer). Run-on Oligomerization, a novel strategy to control enzyme activity and specificity. FASEB Machines on Genes meeting. Snowmass, Colorado.
• Horton, N. C. (2017, June). DNA Binding and Cleavage by Human Parvovirus B19 NS1 Nuclease Domain. Invitation to speak at University of Jyväskylä, Finland. Jyväskylä, Finland: University of Jyväskylä, Finland.
• Horton, N. C. (2017, May). Regulation of Enzyme Activity via Filament Formation. Seminar at UCLA. Los Angeles, CA: UCLA.
• Horton, N. C. (2017, September). Run-On Oligomerization, a Novel Strategy to Control Enzyme Activity and Specificity. CBC Seminar Series. Tucson, AZ: CBC Department.
• Horton, N. C. (2017, Spring). DNA Binding and Cleavage by Human Parvovirus B19 NS1 Nuclease Domain. University of Kansas Medical Center Kansas City. Kansas City, MO: Kansas City Medical Center.
• Horton, N. C. (2017, Spring). Regulation of Enzyme Activity via Filament Formation. Invitation to speak at Portland State University. Portland, Oregon: Portland State University.
• Horton, N. C. (2016, December). Regulation of Enzyme Activity via Filament Formation. Zing Nucleic Acids Conference. Tampa, Florida: Zing.
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  Presented a talk of my research
• Horton, N. C. (2016, October). Regulation of Enzyme Activity via Filament Formation. BCP journal club. Tucson, AZ: Biological Chemistry Graduate Program.
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  Presented a talk to the BCP graduate students.
• Horton, N. C. (2016, Summer). Regulation of Enzyme Activity via Filament Formation. Machines on Genes Conference. Mecclesfield, UK: FASEB.
• Horton, N. C., & Sanchez, J. (2016, Fall). Presentation by my graduate student Jonathan Sanchez. Zing Nucleic Acids. Tampa, FL: Zing.
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  My student presented our work at a national research conference.
• Horton, N. C. (2015, April 2015). Allosteric Modulation of DNA Cleavage and Sequence Specificity by Run-On Oligomerization. Presentation to Department of Chemistry, SDSU. San Diego State University, San Diego, California: San Diego State University.
• Horton, N. C. (2015, August 2015). Allosteric Modulation of DNA Cleavage and Sequence Specificity by Run-On Oligomerization. New England Biolabs Meeting. Gdansk, Poland.
• Horton, N. C. (2015, March 17, 2015). Allosteric Modulation of DNA Cleavage and Sequence Specificity by Run-On Oligomerization. West Coast Protein Crystallography Workshop. Monterey, California.
• Horton, N. C. (2015, September 17). Allosteric Modulation of DNA Cleavage and Sequence Specificity by Run-On Oligomerization. Invitation to speak at New Mexico State University as part of the Chemistry and Biochemistry Colloquim. Los Cruces, New Mexico: Chemistry & Biochemistry Department, NMSU.

Poster Presentations

• Horton, N., Park, C., Shah, S., Stewart, A., Talley, H., Lyumkis, D., Potter, C., Carragher, B., Ma, X., & Wysocki, V. (2013, June). Allosteric Regulation of DNA Cleavage and Sequence-Specificity via Run-on Oligomerization. Nucleic Acids Gordon Research ConferenceUniversity of New England.
• Horton, N., Park, C., Shah, S., Stewart, A., Ma, X., Wysocki, V., Jacovety, E., Talley, H., Agrawal, A., & Bitinaite, J. (2012, April). Activation of DNA Cleavage by Oligomerization of a Sequence Specific Endonuclease. American Society of Biochemists and Molecular Biologists Annual Meeting. San Diego, CA.

Others

• Horton, N., Sam, M., Connolly, B., & Perona, J. (1999, JAN). Structural mechanism of phosphoryl transfer in EcoRV restriction endonuclease. BIOPHYSICAL JOURNAL.