Lisa F Rezende

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Courses

Scholarly Contributions

Chapters

• Dean, M., Friedman, S., Sutphen, R., Clark, B. A., Duquette, D., & Rezende, L. F. (2021). Partners, Not Participants: Engaging Patients in the American BRCA Outcomes and Utilization of Testing (ABOUT) Network. In Researching Health Together: Engaging Patients and Stakeholders, From Topic Identification to Policy Change. doi:10.4135/9781071909539.n10
• Dean, M., Friedman, S. J., Sutphen, R., Bourquardez Clark, E., Duquette, D., & Rezende, L. F. (2020). Partners, not participants: Engaging patients in the American BRCA outcomes and utilization of testing (ABOUT) network. In Patient and stakeholder involvement in health research. Sage.

Journals/Publications

• Rezende, L., Yi, R. H., Welcsh, P., Dearfield, C. T., Owens, K., & Friedman, S. J. (2022). Effectiveness of a tool to increase understanding of breast cancer media articles. Health Education Journal, 001789692211458. doi:10.1177/00178969221145802
• Blowers, P., Elfring, L. K., Talanquer, V. A., Rezende, L. F., Eadie, E. C., Maximillian, J., Kim, Y. A., Southard, K. M., Southard, K. M., Kim, Y. A., Maximillian, J., Eadie, E. C., Rezende, L. F., Talanquer, V. A., Elfring, L. K., & Blowers, P. (2021). Responsive Teaching in Online Learning Environments: Using an Instructional Team to Promote Formative Assessment and Sense of Community. Journal of College Science Teaching, 50(4).
• Pugh Yi, R. H., Rezende, L. F., Deerfield, C. T., Welcsh, P. L., & Friedman, S. J. (2019). Results of a Pilot Test of Effects of an Online Resource on Lay Audience Understanding of Media Reports on Breast Cancer Research. Health Education Journal, 78(5), 607-617.
• Rezende, L., Yi, R. H., Dearfield, C. T., Welcsh, P., & Friedman, S. J. (2019). Results of a pilot test of effects of an online resource on lay audience understanding of media reports on breast cancer research. Health Education Journal, 78(5), 607-617. doi:10.1177/0017896919841406
• Bolger, M. S., Rezende, L. F., Dykstra, E. M., Elfring, L. K., Katcher, J., Nadler, M., & Hester, S. D. (2018). Authentic Inquiry through Modeling in Biology (AIM-Bio): An Introductory Laboratory Curriculum that Increases Undergraduates' Scientific Agency and Skills.. CBE: Life Science Education, 17(4), ar63.
• Hester, S. D., Nadler, M., Katcher, J., Elfring, L. K., Dykstra, E., Rezende, L. F., & Bolger, M. S. (2018). Authentic Inquiry through Modeling in Biology (AIM-Bio): An Introductory Laboratory Curriculum That Increases Undergraduates' Scientific Agency and Skills. CBE life sciences education, 17(4), ar63.
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  Providing opportunities for science, technology, engineering, and mathematics undergraduates to engage in authentic scientific practices is likely to influence their view of science and may impact their decision to persist through graduation. Laboratory courses provide a natural place to introduce students to scientific practices, but existing curricula often miss this opportunity by focusing on confirming science content rather than exploring authentic questions. Integrating authentic science within laboratory courses is particularly challenging at high-enrollment institutions and community colleges, where access to research-active faculty may be limiting. The Authentic Inquiry through Modeling in Biology (AIM-Bio) curriculum presented here engages students in authentic scientific practices through iterative cycles of model generation, testing, and revision. AIM-Bio university and community college students demonstrated their ability to propose diverse models for biological phenomena, formulate and address hypotheses by designing and conducting experiments, and collaborate with classmates to revise models based on experimental data. Assessments demonstrated that AIM-Bio students had an enhanced sense of project ownership and greater identification as scientists compared with students in existing laboratory courses. AIM-Bio students also experienced measurable gains in their nature of science understanding and skills for doing science. Our results suggest AIM-Bio as a potential alternative to more resource-intensive curricula with similar outcomes.
• Rezende, L. F., Elfring, L. K., Hester, S. D., Nadler, M., Katcher, J., Dykstra, E., & Bolger, M. S. (2018). Authentic Inquiry through Modeling in Biology (AIM-Bio): An Introductory Laboratory Curriculum That Increases Undergraduates’ Scientific Agency and Skills. CBE—Life Sciences Education, 17(4), ar63. doi:10.1187/cbe.18-06-0090
• Samimi, G., Bernardini, M. Q., Brody, L. C., Caga-Anan, C. F., Campbell, I. G., Chenevix-Trench, G., Couch, F. J., Dean, M., de Hullu, J. A., Domchek, S. M., Drapkin, R., Spencer Feigelson, H., Friedlander, M., Gaudet, M. M., Harmsen, M. G., Hurley, K., James, P. A., Kwon, J. S., Lacbawan, F., , Lheureux, S., et al. (2017). Traceback: A Proposed Framework to Increase Identification and Genetic Counseling of BRCA1 and BRCA2 Mutation Carriers Through Family-Based Outreach. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 35(20), 2329-2337.
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  In May 2016, the Division of Cancer Prevention and the Division of Cancer Control and Population Sciences, National Cancer Institute, convened a workshop to discuss a conceptual framework for identifying and genetically testing previously diagnosed but unreferred patients with ovarian cancer and other unrecognized BRCA1 or BRCA2 mutation carriers to improve the detection of families at risk for breast or ovarian cancer. The concept, designated Traceback, was prompted by the recognition that although BRCA1 and BRCA2 mutations are frequent in women with ovarian cancer, many such women have not been tested, especially if their diagnosis predated changes in testing guidelines. The failure to identify mutation carriers among probands represents a lost opportunity to prevent cancer in unsuspecting relatives through risk-reduction intervention in mutation carriers and to provide appropriate reassurances to noncarriers. The Traceback program could provide an important opportunity to reach families from racial, ethnic, and socioeconomic groups who historically have not sought or been offered genetic counseling and testing and thereby contribute to a reduction in health disparities in women with germline BRCA mutations. To achieve an interdisciplinary perspective, the workshop assembled international experts in genetics, medical and gynecologic oncology, clinical psychology, epidemiology, genomics, cost-effectiveness modeling, pathology, bioethics, and patient advocacy to identify factors to consider when undertaking a Traceback program. This report highlights the workshop deliberations with the goal of stimulating research and providing a framework for pilot studies to assess the feasibility and ethical and logistical considerations related to the development of best practices for implementation of Traceback studies.
• Yi, R. H., Rezende, L. F., Huynh, J., Kramer, K., Cranmer, M., Schlager, L., Dearfield, C. T., & Friedman, S. J. (2017). XRAYS (eXamining Relevance of Articles to Young Survivors) Program Survey of Information Needs and Media Use by Young Breast Cancer Survivors and Young Women at High-Risk for Breast Cancer. Health communication, 1-6.
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  Women age 45 years or younger with breast cancer, or who are at high-risk for breast cancer due to previously having the disease or to genetic risk, have distinct health risks and needs from their older counterparts. Young women frequently seek health information through the Internet and mainstream media, but often find it does not address their particular concerns, that it is difficult to evaluate or interpret, or even misleading. To help women better understand media coverage about new research, Facing Our Risk of Cancer Empowered (FORCE) developed the CDC-funded XRAYS (eXamining Relevance of Articles to Young Survivors) program. To assure that the XRAYS program is responsive to the community's needs, FORCE launched a web-based survey to assess where young women seek information about breast cancer, and to learn their unmet information needs. A total of 1,178 eligible women responded to the survey. In general, the breast cancer survivors and high-risk women between ages 18-45 years who responded to this survey, are using multiple media sources to seek information about breast cancer risk, prevention, screening, and treatment. They place trust in several media sources and use them to inform their medical decisions. Only about one-third of respondents to this survey report discussing media sources with their health care providers. Current survey results indicate that, by providing credible information on the quality of evidence and reporting in media reports on cancer, XRAYS is addressing a key need for health information. Results suggest that it will be useful for XRAYS to offer reviews of articles on a broad range of topics that can inform decisions at each stage of risk assessment and treatment.
• Tran, N. Q., Rezende, L. F., Qimron, U., Richardson, C. C., & Tabor, S. (2008). Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine. Proceedings of the National Academy of Sciences of the United States of America, 105(27), 9373-8.
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  Bacteriophage T7 DNA polymerase efficiently incorporates dideoxynucleotides into DNA, resulting in chain termination. Dideoxythymidine (ddT) present in the medium at levels not toxic to Escherichia coli inhibits phage T7. We isolated 95 T7 phage mutants that were resistant to ddT. All contained a mutation in T7 gene 1.7, a nonessential gene of unknown function. When gene 1.7 was expressed from a plasmid, T7 phage resistant to ddT still arose; analysis of 36 of these mutants revealed that all had a single mutation in gene 5, which encodes T7 DNA polymerase. This mutation changes tyrosine-526 to phenylalanine, which is known to increase dramatically the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. DNA synthesis in cells infected with wild-type T7 phage was inhibited by ddT, suggesting that it resulted in chain termination of DNA synthesis in the presence of gene 1.7 protein. Overexpression of gene 1.7 from a plasmid rendered E. coli cells sensitive to ddT, indicating that no other T7 proteins are required to confer sensitivity to ddT.
• Tran, N. Q., Tabor, S., Rezende, L. F., Qimron, U., & Richardson, C. C. (2008). Gene 1.7 of Bacteriophage T7 Confers Sensitivity of Phage Growth to Dideoxythymidine. Journal of Biological Chemistry. doi:10.1096/fasebj.22.1_supplement.651.5
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  Bacteriophage T7 has long been used as a model for studying DNA replication. One of the interesting features of this system is the ability of T7 DNA polymerase to efficiently incorporate dideoxynucleotides into DNA, resulting in chain termination. We show that dideoxythymidine (ddT) present in the media at levels not toxic to the host Escherichia coli results in inhibition of phage T7 growth. We have isolated 100 T7 phage that are resistant to ddT. Surprisingly, all contain a mutation in T7 gene 1.7, a nonessential gene of unknown function. When T7 gene 1.7 is expressed from a plasmid, suppressor T7 phage that are resistant to ddT still arise; analysis of 36 of these mutants reveals that all have mutations in gene 5, that encodes the T7 DNA polymerase. The strongest suppressors change tyrosine 526 of gene 5 to phenylalanine. This mutation is known to dramatically increase the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. These results suggest that in the presence of the T7 gene 1.7 protein, ddT is inhibitory to phage T7 because it acts as a chain terminator for DNA synthesis. In support of this, whereas DNA synthesis in E. coli infected with wild-type T7 is inhibited by ddT, no inhibition is observed in the absence of gene 1.7. Overexpression of gene 1.7 from a plasmid renders E. coli cells sensitive to ddT in the absence of T7 phage, indicating that gene 1.7 protein does not require other T7 proteins to confer sensitivity to ddT. We propose that gene 1.7 protein plays a critical role in the conversion of ddT to ddTTP.
• Rezende, L. F., & Prasad, V. R. (2004). Nucleoside-analog resistance mutations in HIV-1 reverse transcriptase and their influence on polymerase fidelity and viral mutation rates. The international journal of biochemistry & cell biology, 36(9), 1716-34.
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  Nucleoside-analog inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) were the first drugs used against the virus. It is long known that monotherapy with these and other drugs leads to the rapid development of viral resistance and it is being increasingly appreciated that a significant percentage of individuals receiving highly active antiretroviral therapy (HAART) also develop resistance. Considering the fact that RT is responsible both for optimal rate of replication and an accurate copying of the viral genome, the consequence of drug-resistance mutations in RT to the biochemistry of this enzyme and to the biology of the virus are critically important. The biochemistry of HIV-1 reverse transcriptase variants harboring nucleoside-analog resistance mutations has been studied extensively. In this review, we describe a number of studies into the polymerase fidelity of nucleoside-analog resistant HIV-1 reverse transcriptase as well as the mutation rate of HIV-1 harboring these mutations.
• He, Z. G., Rezende, L. F., Willcox, S., Griffith, J. D., & Richardson, C. C. (2003). The Carboxyl-terminal Domain of Bacteriophage T7 Single-stranded DNA-binding Protein Modulates DNA Binding and Interaction with T7 DNA Polymerase. Journal of Biological Chemistry. doi:10.1074/jbc.m304318200
• He, Z. G., Rezende, L. F., Willcox, S., Griffith, J. D., & Richardson, C. C. (2003). The carboxyl-terminal domain of bacteriophage T7 single-stranded DNA-binding protein modulates DNA binding and interaction with T7 DNA polymerase. The Journal of biological chemistry, 278(32), 29538-45.
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  Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein (gp2.5). Previous studies have demonstrated that the acidic carboxyl terminus of the protein is essential and that it mediates multiple protein-protein interactions. A screen for lethal mutations in gene 2.5 uncovered a variety of essential amino acids, among which was a single amino acid substitution, F232L, at the carboxyl-terminal residue. gp2.5-F232L exhibits a 3-fold increase in binding affinity for single-stranded DNA and a slightly lower affinity for T7 DNA polymerase when compared with wild type gp2.5. gp2.5-F232L stimulates the activity of T7 DNA polymerase and, in contrast to wild-type gp2.5, promotes strand displacement DNA synthesis by T7 DNA polymerase. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta 26C, binds single-stranded DNA 40-fold more tightly than the wild-type protein and cannot physically interact with T7 DNA polymerase. gp2.5-Delta 26C is inhibitory for DNA synthesis catalyzed by T7 DNA polymerase on single-stranded DNA, and it does not stimulate strand displacement DNA synthesis at high concentration. The biochemical and genetic data support a model in which the carboxyl-terminal tail modulates DNA binding and mediates essential interactions with T7 DNA polymerase.
• Hyland, E. M., Rezende, L. F., & Richardson, C. C. (2003). The DNA binding domain of the gene 2.5 single-stranded DNA-binding protein of bacteriophage T7. The Journal of biological chemistry, 278(9), 7247-56.
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  Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA-binding protein that is essential for viral survival. Its crystal structure reveals a conserved oligosaccharide/oligonucleotide binding fold predicted to interact with single-stranded DNA. However, there is no experimental evidence to support this hypothesis. Recently, we reported a genetic screen for lethal mutations in gene 2.5 that we are using to identify functional domains of the gene 2.5 protein. This screen uncovered a number of mutations that led to amino acid substitutions in the proposed DNA binding domain. Three variant proteins, gp2.5-Y158C, gp2.5-K152E, and gp2.5-Y111C/Y158C, exhibit a decrease in binding affinity for oligonucleotides. A fourth, gp2.5-K109I, exhibits an altered mode of binding single-stranded DNA. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta26C, binds single-stranded DNA 10-fold more tightly than the wild-type protein. The three altered proteins defective in single-stranded DNA binding cannot mediate the annealing of homologous DNA, whereas gp2.5-Delta26C mediates the reaction more effectively than does wild-type. Gp2.5-K109I retains this annealing ability, albeit slightly less efficiently. With the exception of gp2.5-Delta26C, all variant proteins form dimers in solution and physically interact with T7 DNA polymerase.
• Rezende, L. F., Willcox, S., Griffith, J. D., & Richardson, C. C. (2003). A Single-stranded DNA-binding Protein of Bacteriophage T7 Defective in DNA Annealing. Journal of Biological Chemistry. doi:10.17615/ww9e-eg80
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  The annealing of complementary strands of DNA is a vital step during the process of DNA replication, recombination, and repair. In bacteriophage T7-infected cells, the product of viral gene 2.5, a single-stranded DNA-binding protein, performs this function. We have identified a single amino acid residue in gene 2.5 protein, arginine 82, that is critical for its DNA annealing activity. Expression of gene 2.5 harboring this mutation does not complement the growth of a T7 bacteriophage lacking gene 2.5. Purified gene 2.5 protein-R82C binds single-stranded DNA with a greater affinity than the wild-type protein but does not mediate annealing of complementary strands of DNA. A carboxyl-terminal-deleted protein, gene 2.5 protein-Δ26C, binds even more tightly to single-stranded DNA than does gene 2.5 protein-R82C, but it anneals homologous strands of DNA as well as does the wild-type protein. The altered protein forms dimers and interacts with T7 DNA polymerase comparable with the wild-type protein. Gene 2.5 protein-R82C condenses single-stranded M13 DNA in a manner similar to wild-type protein when viewed by electron microscopy. The annealing of complementary strands of DNA is a vital step during the process of DNA replication, recombination, and repair. In bacteriophage T7-infected cells, the product of viral gene 2.5, a single-stranded DNA-binding protein, performs this function. We have identified a single amino acid residue in gene 2.5 protein, arginine 82, that is critical for its DNA annealing activity. Expression of gene 2.5 harboring this mutation does not complement the growth of a T7 bacteriophage lacking gene 2.5. Purified gene 2.5 protein-R82C binds single-stranded DNA with a greater affinity than the wild-type protein but does not mediate annealing of complementary strands of DNA. A carboxyl-terminal-deleted protein, gene 2.5 protein-Δ26C, binds even more tightly to single-stranded DNA than does gene 2.5 protein-R82C, but it anneals homologous strands of DNA as well as does the wild-type protein. The altered protein forms dimers and interacts with T7 DNA polymerase comparable with the wild-type protein. Gene 2.5 protein-R82C condenses single-stranded M13 DNA in a manner similar to wild-type protein when viewed by electron microscopy. Gene 2.5 protein is required for bacteriophage T7 growth (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (60) Google Scholar). Gene 2.5 protein acts as a nonspecific single-stranded DNA (ssDNA) 1The abbreviations used are: ssDNA, single-stranded DNA; WT, wild-type; NTA, nitrilotriacetic acid; SSB protein, single-stranded binding protein.-binding protein, binding ssDNA preferentially over double-stranded DNA (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar). ssDNA binding proteins participate in multiple steps of DNA replication, recombination, and repair (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (60) Google Scholar, 2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar, 3Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 4Chase J.W. Williams K.R. Annu. Rev. Biochem. 1986; 55: 103-136Crossref PubMed Scopus (448) Google Scholar, 5Reuben R.C. Gefter M.L. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 1846-1850Crossref PubMed Scopus (49) Google Scholar, 6Scherzinger E. Litfin F. Jost E. Mol. Gen. Genet. 1973; 123: 247-262Crossref PubMed Scopus (40) Google Scholar, 7Araki H. Ogawa H. Virology. 1981; 111: 509-515Crossref PubMed Scopus (18) Google Scholar, 8Araki H. Ogawa H. Mol. Gen. Genet. 1981; 183: 66-73Crossref PubMed Scopus (15) Google Scholar, 9Nakai H. Richardson C.C. J. Biol. Chem. 1988; 263: 9831-9839Abstract Full Text PDF PubMed Google Scholar, 10Kong D. Richardson C.C. EMBO J. 1996; 15: 2010-2019Crossref PubMed Scopus (52) Google Scholar, 11Kong D. Nossal N.G. Richardson C.C. J. Biol. Chem. 1997; 272: 8380-8387Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 12Lee J. Chastain 2nd, P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 13Yu M. Masker W. J. Bacteriol. 2001; 183: 1862-1869Crossref PubMed Scopus (13) Google Scholar). Whereas gene 2.5 protein is functionally equivalent to Escherichia coli SSB protein and the bacteriophage T4 gene 32 protein, it lacks significant sequence homology to these proteins, and neither of these proteins can replace its function in vivo (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (60) Google Scholar). In addition, gene 2.5 protein binds ssDNA with a lower affinity than either the E. coli or T4 proteins (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar). Gene 2.5 protein also physically and functionally interacts with T7 DNA polymerase and T7 gene 4 product, a primase/helicase (3Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 9Nakai H. Richardson C.C. J. Biol. Chem. 1988; 263: 9831-9839Abstract Full Text PDF PubMed Google Scholar, 12Lee J. Chastain 2nd, P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). These interactions are mediated by a highly acidic carboxyl-terminal motif and are essential for coordination of leading and lagging strand DNA synthesis in vitro (12Lee J. Chastain 2nd, P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 14Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar, 15Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In addition to binding ssDNA and physically interacting with T7 DNA polymerase, gene 2.5 protein also facilitates the annealing of complementary strands of DNA (10Kong D. Richardson C.C. EMBO J. 1996; 15: 2010-2019Crossref PubMed Scopus (52) Google Scholar, 11Kong D. Nossal N.G. Richardson C.C. J. Biol. Chem. 1997; 272: 8380-8387Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar). Homologous DNA annealing is a vital activity during the process of DNA replication, recombination, and repair (17Iyer L.M. Koonin E.V. Aravind L. BMC Genomics. 2002; 3: 8Crossref PubMed Scopus (144) Google Scholar). A number of proteins have evolved to carry out this vital function, such as the RecA protein (18Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar, 19Bianco P.R. Tracy R.B. Kowalczykowski S.C. Front. Biosci. 1998; 3: 570-603Crossref PubMed Google Scholar) and members of the single strand annealing family that includes the E. coli RecT protein, the Redβ protein from bacteriophage λ, and the eukaryotic annealing protein Rad52 (17Iyer L.M. Koonin E.V. Aravind L. BMC Genomics. 2002; 3: 8Crossref PubMed Scopus (144) Google Scholar, 20Mortensen U.H. Bendixen C. Sunjevaric I. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10729-10734Crossref PubMed Scopus (390) Google Scholar, 21Hall S.D. Kane M.F. Kolodner R.D. J. Bacteriol. 1993; 175: 277-287Crossref PubMed Google Scholar, 22Kmiec E. Holloman W.K. J. Biol. Chem. 1981; 256: 12636-12639Abstract Full Text PDF PubMed Google Scholar, 23Reddy G. Golub E.I. Radding C.M. Mutat. Res. 1997; 377: 53-59Crossref PubMed Scopus (85) Google Scholar). Unlike the RecA protein, the gene 2.5 protein does not require ATP (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar), and it cannot mediate strand transfer (11Kong D. Nossal N.G. Richardson C.C. J. Biol. Chem. 1997; 272: 8380-8387Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar). Gene 2.5 protein bears some similarity to the RecT protein and its family members, proteins that also mediate DNA annealing in an ATP-independent fashion (17Iyer L.M. Koonin E.V. Aravind L. BMC Genomics. 2002; 3: 8Crossref PubMed Scopus (144) Google Scholar). Structurally, gene 2.5 protein differs from members of this family, which form multimeric ring structures in the presence and absence of ssDNA (24Kagawa W. Kurumizaka H. Ishitani R. Fukai S. Nureki O. Shibata T. Yokoyama S. Mol. Cell. 2002; 10: 359-371Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 25Singleton M.R. Wentzell L.M. Liu Y. West S.C. Wigley D.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13492-13497Crossref PubMed Scopus (176) Google Scholar, 26Passy S.I. Yu X. Li Z. Radding C.M. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4279-4284Crossref PubMed Scopus (84) Google Scholar). Gene 2.5 protein, on the other hand, is a dimer in solution (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar), and its three-dimensional structure resembles that of other ssDNA-binding proteins (27Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (83) Google Scholar). Similar to T4 gene 32 protein and E. coli SSB protein, gene 2.5 protein features an oligonucleotide/oligosaccaride binding fold (Fig. 1) (27Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (83) Google Scholar). Although both T4 gene 32 protein and E. coli SSB protein have been shown to mediate DNA annealing (28Christiansen C. Baldwin R.L. J. Mol. Biol. 1977; 115: 441-454Crossref PubMed Scopus (57) Google Scholar, 29Alberts B.M. Frey L. Nature. 1970; 227: 1313-1318Crossref PubMed Scopus (448) Google Scholar), T7 gene 2.5 protein does so much more efficiently (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar). The biochemical basis of the efficient DNA annealing activity of gene 2.5 protein is unknown. It seems likely that it involves interactions between two gene 2.5 protein-coated ssDNA molecules. A previous study has shown that the ability to bind ssDNA is critical for this reaction to occur (30Hyland E.M. Rezende L.F. Richardson C.C. J. Biol. Chem. 2003; 278: 7247-7256Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). It is also possible that interactions at the dimer interface are involved in this process. Two gene 2.5 proteins with alterations in the dimer interface retained the ability to mediate DNA annealing, in a manner similar to the WT protein, whereas a third did so in a slightly longer time period (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). We have recently employed a genetic screen to identify functional domains in gene 2.5 protein (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). One of the alterations uncovered by the screen mapped to a loop connecting the prominent α-helix to the β-barrel portion of the structure (Fig. 1). The exact residue, Arg-82, resides in a disordered region of the structure. Here we describe the purification and characterization of this protein and show that it is defective in DNA annealing. Bacterial Strains and Phage—E. coli HMS262 (F– hsdR pro leu – lac – thi – supE tonA – trxA –) and E. coli HMS 89 (xth1 thi – argE mtl – xyl – str – R ara – his – galK lacY proA leu – thr – tsx – supE) were used as hosts for phage experiments. E. coli BL21 (DE3) (F– ompT hsdSB(rB-mB-) gal – dcm – (DE3)) (Novagen) was used to express gene 2.5. T7Δ2.5 phage used in the in vivo DNA synthesis experiments was provided by Jaya Kumar (Harvard Medical School). Plasmids, Oligonucleotides, and Proteins—The following oligonucleotides were purchased from Oligos Etc.: T72.5NdeI (5′-CGTAGGATCCATATGGCTAAGAAGATTTTCACCTC-3′), T72.5BamH1 (5′-CGTAGGATCCACTTAGAAGTCTCCGTC-3′), and Oligo 70 (5′-GACCATATCCTCCACCCTCCCCAATATTGACCATCAACCCTTCAC CTCACTTCACTCCACTATACCACTC-3′). The oligonucleotide BCMP206 (5′-TAACGCCAGGGTTTTCCCAGTCACG-3′) was synthesized by the Biopolymer Laboratory, Harvard Medical School. M13 (mGP1-2) DNA and T7 DNA polymerase lacking exonuclease activity (30Hyland E.M. Rezende L.F. Richardson C.C. J. Biol. Chem. 2003; 278: 7247-7256Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) were kindly provided by Stanley Tabor (Harvard Medical School). Gene 2.5 protein-Δ26C was provided by Edel Hyland (Harvard Medical School). His-gene 2.5 protein-Δ26C was provided by James Stattel (Harvard Medical School). T7 DNA polymerase was provided by Donald Johnson and Joon-Soo Lee (Harvard Medical School). Purification of WT gene 2.5 protein and His-gene 2.5 protein was described previously (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). E. coli SSB protein was purchased from U.S. Biochemical Corp. All other proteins were purified as described below. In Vivo DNA Synthesis Assay—Phage DNA synthesis was determined as described previously (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). E. coli HMS262 cells transformed with pETGP2.5-R82C were grown in Davis minimal media supplemented with ampicillin at 30 °C. Cells were infected with T7Δ2.5 phage at a multiplicity of infection of 7. At 5-min intervals postinfection, 200-μl samples were removed. [3H]thymidine (50μCi/ml) was added, and after a 90-s incubation at 30 °C, 40 μl of an ice-cold solution of 50 mm Tris-HCl (pH 7.5), 2 mm EDTA, 2% SDS was added to the sample. The lysed cells were then spotted onto DE81 filters, washed, and air-dried. [3H]Thymidine incorporation into DNA was then measured by liquid scintillation counting. Protein Purification—Gene 2.5 protein-R82C was overproduced and purified using a procedure previously employed to purify WT gene 2.5 protein (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). A 1-liter culture of E. coli BL21(DE3) (Novagen) expressing gene 2.5 protein-R82C was grown, and gene 2.5 protein-R82C was purified by precipitation in polyethyleneimine (pH 7.5), followed by fractionation on an HQ column and a gel filtration column. The protein was 99% pure as determined by denaturing polyacrylamide gel electrophoresis followed by Coomassie Blue staining and was free of contaminating deoxyribonuclease activity (data not shown). Protein concentrations were calculated from UV spectrophotometer readings at 280 mm, using the calculated extinction coefficient at 280 nm of 2.59 × 104 M–1 cm–1 for gene 2.5 protein-R82C (32Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5072) Google Scholar). His-tagged gene 2.5 protein-R82C was purified using a previously described method (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Determining DNA Binding Affinity by Electrophoretic Mobility Shift Assay—The ssDNA binding activity of gene 2.5 protein was assessed by a electrophoretic mobility shift assay (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Gene 2.5 proteins (diluted in a buffer of 20 mm Tris (pH 7.5), 10 mm β-mercaptoethanol, and 500 μg/ml bovine serum albumin) were incubated with 3 nm33P-end-labeled 70-mer oligonucleotide, 15 mm MgCl2, 5 mm dithiothreitol, 50 mm KCl, 10% glycerol, 0.01% bromophenyl blue. ssDNA was separated from ssDNA-protein complex on 10% TBE Ready Gels (Bio-Rad) running in 0.5× Tris/glycine buffer (12.5 mm Tris base, 95 mm glycine, 0.5 mm EDTA). Gels were dried and exposed to a Fujix phosphor imager plate, and the amount of radioactivity was calculated using ImageQuant software. DNA Annealing Assay—The ability of WT gene 2.5 protein to mediate the annealing of homologous strands of DNA was assessed using an in vitro annealing assay developed by Tabor and Richardson (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar). A 310-nucleotide internally labeled ssDNA fragment was generated as described previously (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar, 31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). DNA annealing was assayed in reactions containing 4 nm32P-labeled ssDNA fragment, 20 μm M13 mGP1-2 ssDNA, 20 mm Tris-Cl (pH 7.5), 1 mm dithiothreitol, 10 mm MgCl2, 50 mm NaCl, and various concentrations of gene 2.5 protein. Unless noted otherwise, reactions were incubated at 30 °C for 8 min. Time course experiments were carried out at 30 °C with 10 μm gene 2.5 protein, and the reaction was stopped by the addition of SDS to a final concentration of 0.5%. Reaction products were analyzed on a 0.8% agarose gel at 75 V for 1 h at room temperature, dried under vacuum, and exposed to a Fujix phosphor imager plate, and radioactivity was calculated using ImageQuant software. Plots of the data represent the background-corrected average of three experiments. Electron Microscopy—WT and altered gene 2.5 proteins or E. coli SSB protein were diluted to 500 ng/μl in 20 mm Hepes/NaOH, (pH 7.5), 20% glycerol, mixed with single-stranded WT M13 DNA at 25 ng/μl in a buffer containing 10 mm Hepes/NaOH (pH 7.5), 50 mm NaCl final concentration. MgCl2 was added to the reaction buffer to 10 mm where indicated. Binding reactions with protein/DNA ratios (μg/μg) ranging from 40:1 for WT gene 2.5 protein to 10:1 for mutants and E. coli SSB protein were incubated for 15 min at room temperature in a 50-μl total reaction volume. Following the binding reactions, the samples were fixed with an equal volume of 1.2% glutaraldehyde for 5 min at room temperature and then loaded onto a 2-ml column of Bio-Gel A-5m previously equilibrated in 10 mm Tris·HCl (pH 7.5), 0.5 mm EDTA. The same buffer was then used to elute the sample from the column and 250-μl fractions were collected. Aliquots of the protein-DNA containing fractions were mixed with a buffer containing spermidine (33Griffith J.D. Christiansen G. Annu. Rev. Biophys. Bioeng. 1978; 7: 19-35Crossref PubMed Scopus (181) Google Scholar) for 3 s and quickly applied to a mesh copper grid coated with a thin carbon film, glow-charged shortly before sample application. Following adsorption of the samples to the electron microscopy support for 1–2 min, the grids were subjected to a dehydration procedure in which the water content of the wash solutions was gently replaced by a serial increase in ethanol concentration to 100% and then air-dried. The samples were then rotary shadowcast with tungsten at 10–7 torr and examined in a Philips CM 12 TEM instrument at 40 kV. Micrographs, taken at × 46,000, were scanned using a Nikon LS-4500AF film scanner, and panels were arranged using Adobe Photoshop. Gel Filtration Analysis—Gel filtration analysis was performed as previously described (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Fifty μg of gene 2.5 protein-R82C diluted in buffer S (final concentration 4 μm) were loaded on a Superdex 75 column (Amersham Biosciences). A standard curve of K av versus log M r was generated by applying low molecular weight protein standards (Amersham Biosciences) to the column under the same conditions. Analysis of Protein-Protein Interaction by Surface Plasmon Resonance—The interaction between gene 2.5 protein and T7 DNA polymerase was measured by SPR using the BIACORE 3000 system as described previously (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Briefly, 10 μl of 500 nm histidine-tagged gene 2.5 protein, gene 2.5 protein-R82C, and gene 2.5 protein-Δ26C were immobilized onto separate lanes of a nickel-charged sensor chip NTA (BIAcore). This amount of protein correlated to ∼7,000 resonance units. Ten μl of 500 nm T7 DNA polymerase or bovine serum albumin were passed over the chip, and dissociation of T7 DNA polymerase was monitored for 10 min while passing 100 μl of running buffer over the chip. Each analysis was performed in triplicate and repeated on three separate days. The kinetics of the gene 2.5 protein-T7 DNA polymerase interaction was assessed by binding 50 nm of either WT or mutant histidine-tagged gene 2.5 protein to the nickel-charged chip and then passing 10 μl of 0–50 nm T7 DNA over the chip. BIAevaluation software was used to determine dissociation constants (K D), which were solved using the simultaneous k a/k d data fit. Gene 2.5 Protein-R82C Cannot Support T7 DNA Synthesis or T7 Phage Growth—Gene 2.5 is essential for the growth of bacteriophage T7 (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (60) Google Scholar). In this study, we examine a mutation, leading to a single amino acid change, arginine 82 to cysteine, that was isolated as part of a screen for lethal mutations in gene 2.5 (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). As such, it was unable to support the growth of T7 phage lacking gene 2.5 (T7Δ2.5) (Table I). Interestingly, this mutation does not affect the function of WT gene 2.5 protein based on the fact that expression of the mutated gene from a plasmid does not inhibit the growth of WT T7 phage (Table I).Table IPlating efficiency of T7 and T7Δ2.5 on E. coli strains containing plasmids expressing WT or mutant T7 gene 2.5 proteinsPlasmidT7T7Δ2.5pETGP2.511pETGP2.5-R82C0.852.0 × 10-5 Open table in a new tab Since gene 2.5 is an essential gene and its product is involved in DNA synthesis in vitro, we examined the ability of gene 2.5 protein-R82C to carry out DNA synthesis in vivo. E. coli cells expressing the WT or mutant gene 2.5 protein were grown to midlog phase and then infected with a T7 phage lacking gene 2.5. At specific time points, aliquots of cells were removed and mixed with radioactively labeled thymidine. After 90 s, the reactions were terminated. Results of such an experiment are shown in Fig. 2. DNA synthesis peaks ∼30 min after infection in cells expressing WT gene 2.5. As a control, no DNA synthesis is observed in cells harboring gene 2.5 lacking the coding sequence for the carboxyl-terminal motif (gene 2.5 protein-Δ26C). Similarly, DNA synthesis declines soon after infection in cells expressing gene 2.5 protein-R82C. Therefore, it is likely that this mutant is lethal because it is defective in some aspect of DNA metabolism. Gene 2.5 Protein-R82C Binds ssDNA—One of the primary attributes of gene 2.5 protein is its ability to bind ssDNA (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar). In the current study, we assessed the ability of the altered gene 2.5 proteins to bind ssDNA using an electrophoretic mobility shift assay. Using this method, we previously calculated the dissociation constant (K D) for WT gene 2.5 protein to be 2.6 × 10–6m (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). As shown in Fig. 3, the mobility of a 70-mer oligonucleotide is retarded as increasing amounts of gene 2.5 protein-R82C are added. Gene 2.5 protein-R82C binds ssDNA with ∼10-fold higher affinity than does the WT protein (K D = 3.0 × 10–7m). Thus, the amino acid alteration causes the protein to bind ssDNA with a higher affinity than WT gene 2.5 protein. Since gene 2.5 protein-R82C retains this vital function, we consider it unlikely that the alteration results in a mis-folded protein. Like other ssDNA binding proteins, WT gene 2.5 protein binds ssDNA with a much higher affinity than double-stranded DNA (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar). We examined the binding of gene 2.5 protein-R82C to double-stranded DNA using the electrophoretic mobility shift assay. Gene 2.5 protein-R82C bound a double-stranded 70-base pair DNA weakly and in a manner similar to the WT protein (data not shown). Thus, whereas the alteration, arginine 82 to cysteine, conferred higher ssDNA-binding affinity upon gene 2.5 protein, it did not lead to increased double-stranded DNA binding activity. Gene 2.5 Protein-R82C Is Defective in DNA Annealing—Gene 2.5 protein can anneal homologous strands of ssDNA in vitro (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar, 30Hyland E.M. Rezende L.F. Richardson C.C. J. Biol. Chem. 2003; 278: 7247-7256Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In this study, we looked at the ability of WT and altered gene 2.5 proteins to anneal a 310-nucleotide ssDNA fragment to single-stranded M13 DNA. As previously shown (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar), WT gene 2.5 protein can efficiently anneal these homologous strands of DNA (Fig. 4A). In this reaction, an internally labeled 310-nucleotide ssDNA is mixed with M13 circular ssDNA in the presence of varying concentrations of gene 2.5 protein. The labeled DNA fragment is homologous to a region of the M13 ssDNA. Annealing of the 310-nucleotide fragment to the homologous region of M13 ssDNA does not occur after an 8-min incubation at 30 °C in the absence of gene 2.5 protein (Fig. 4A, lane 1), since we observe a single, rapidly migrating radioactively labeled species on an agarose gel. When the concentration of gene 2.5 protein in the reaction is increased, annealing of the DNA strands begins to occur. In Fig. 4A, lane 4, we observe two species, the faster migrating corresponding to the unannealed 310-nucleotide fragment and a more slowly migrating species corresponding to the annealed product. The more slowly migrating species is present even after extraction with phenol chloroform (data not shown), suggesting that the gel shift is due to the increase in size of the annealed product and not a function of gene 2.5 protein binding to the ssDNA. At even higher concentrations (Fig. 4A), all of the labeled fragment is annealed to the M13 circular ssDNA. As previously shown (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar), DNA annealing is not observed under the same conditions when E. coli SSB protein is added to the reaction (Fig. 4B). Instead, a third species that migrates faster than the annealed product and slower than the fragment is observed upon the addition of E. coli SSB protein. Such a gel shift is noted in all protein concentrations tested (Fig. 4B, lanes 2–7). This species migrates more rapidly than the annealed product produced by gene 2.5 protein under the same conditions (Fig. 4B, lane 8). At pH 7.5, DNA annealing by E. coli SSB protein is dependent on the presence of a polyamine (28Christiansen C. Baldwin R.L. J. Mol. Biol. 1977; 115: 441-454Crossref PubMed Scopus (57) Google Scholar). Since we did not include polyamine in our assay, it is not surprising that E. coli SSB protein could not mediate this reaction under the conditions employed in this study. Gene 2.5 protein-R82C is defective in DNA annealing (Fig. 4C). At the highest concentration test (45 μm), only ∼25% of the fragment is converted to annealed product (Fig. 4C, lane 6). Under the same conditions, WT gene 2.5 protein anneals 100% of the fragment at a concentration of 15 μm (Fig. 4A, lane 5). Like E. coli SSB protein, gene 2.5 protein-R82C has a higher affinity for ssDNA than the WT protein. Thus, it is not surprising that we observe the appearance of a band that probably corresponds to a protein-DNA complex as the concentration of gene 2.5 protein-R82C in the reaction is increased (Fig. 4C, lanes 2–6). Next, we compared DNA annealing mediated by the WT protein with annealing mediated by gene 2.5 protein-R82C over a 4-min time period. In Fig. 5, we show that the WT protein anneals nearly all of the labeled fragment in the reaction in less than 3 min. In contrast, when the same concentration of gene 2.5 protein-R82C is added to the reaction, no annealed product is observed over the 4-min time course. Gene 2.5 protein-R82C and E. coli SSB protein both have a higher affinity for ssDNA than WT gene 2.5
• Rezende, L. F., Willcox, S., Griffith, J. D., & Richardson, C. C. (2003). A single-stranded DNA-binding protein of bacteriophage T7 defective in DNA annealing. The Journal of biological chemistry, 278(31), 29098-105.
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  The annealing of complementary strands of DNA is a vital step during the process of DNA replication, recombination, and repair. In bacteriophage T7-infected cells, the product of viral gene 2.5, a single-stranded DNA-binding protein, performs this function. We have identified a single amino acid residue in gene 2.5 protein, arginine 82, that is critical for its DNA annealing activity. Expression of gene 2.5 harboring this mutation does not complement the growth of a T7 bacteriophage lacking gene 2.5. Purified gene 2.5 protein-R82C binds single-stranded DNA with a greater affinity than the wild-type protein but does not mediate annealing of complementary strands of DNA. A carboxyl-terminal-deleted protein, gene 2.5 protein-Delta26C, binds even more tightly to single-stranded DNA than does gene 2.5 protein-R82C, but it anneals homologous strands of DNA as well as does the wild-type protein. The altered protein forms dimers and interacts with T7 DNA polymerase comparable with the wild-type protein. Gene 2.5 protein-R82C condenses single-stranded M13 DNA in a manner similar to wild-type protein when viewed by electron microscopy.
• Rezende, L. F., Hollis, T., Ellenberger, T., & Richardson, C. C. (2002). Essential Amino Acid Residues in the Single-stranded DNA-binding Protein of Bacteriophage T7. Journal of Biological Chemistry. doi:10.1074/jbc.m207359200
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  Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein. T7 phage with gene 2.5 deleted can grow only on Escherichia coli cells that express gene 2.5 from a plasmid. This complementation assay was used to screen for lethal mutations in gene 2.5. By screening a library of randomly mutated plasmids encoding gene 2.5, we identified 20 different single amino acid alterations in gene 2.5 protein that are lethalin vivo. The location of these essential residues within the three-dimensional structure of gene 2.5 protein assists in the identification of motifs in the protein. In this study we show that a subset of these alterations defines the dimer interface of gene 2.5 protein predicted by the crystal structure. Recombinantly expressed and purified gene 2.5 protein-P22L, gene 2.5 protein-F31S, and gene 2.5 protein-G36S do not form dimers at salt concentrations where the wild-type gene 2.5 protein exists as a dimer. The basis of the lethality of these mutations in vivo is not known because altered proteins retain the ability to bind single-stranded DNA, anneal complementary strands of DNA, and interact with T7 DNA polymerase. Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein. T7 phage with gene 2.5 deleted can grow only on Escherichia coli cells that express gene 2.5 from a plasmid. This complementation assay was used to screen for lethal mutations in gene 2.5. By screening a library of randomly mutated plasmids encoding gene 2.5, we identified 20 different single amino acid alterations in gene 2.5 protein that are lethalin vivo. The location of these essential residues within the three-dimensional structure of gene 2.5 protein assists in the identification of motifs in the protein. In this study we show that a subset of these alterations defines the dimer interface of gene 2.5 protein predicted by the crystal structure. Recombinantly expressed and purified gene 2.5 protein-P22L, gene 2.5 protein-F31S, and gene 2.5 protein-G36S do not form dimers at salt concentrations where the wild-type gene 2.5 protein exists as a dimer. The basis of the lethality of these mutations in vivo is not known because altered proteins retain the ability to bind single-stranded DNA, anneal complementary strands of DNA, and interact with T7 DNA polymerase. single-stranded DNA wild-type dithiothreitol nitrilotriacetic acid surface plasmon resonance Gene 2.5 of bacteriophage T7 is essential for phage growth (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (59) Google Scholar). It encodes a single-stranded DNA (ssDNA)1-binding protein that is functionally similar to the Escherichia coli SSB protein and the gene 32 protein of bacteriophage T4 (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar, 3Chase J.W. Williams K.R. Annu. Rev. Biochem. 1986; 55: 103-136Crossref PubMed Scopus (446) Google Scholar). Like these ssDNA-binding proteins, the gene 2.5 product (wt gene 2.5 protein) is important for DNA replication, recombination, and repair (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (59) Google Scholar, 2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar, 3Chase J.W. Williams K.R. Annu. Rev. Biochem. 1986; 55: 103-136Crossref PubMed Scopus (446) Google Scholar, 4Reuben R.C. Gefter M.L. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 1846-1850Crossref PubMed Scopus (49) Google Scholar, 5Scherzinger E. Litfin F. Jost E. Mol. Gen. Genet. 1973; 123: 247-262Crossref PubMed Scopus (40) Google Scholar, 6Araki H. Ogawa H. Virology. 1981; 111: 509-515Crossref PubMed Scopus (18) Google Scholar, 7Araki H. Ogawa H. Mol. Gen. Genet. 1981; 183: 66-73Crossref PubMed Scopus (15) Google Scholar, 8Nakai H. Richardson C.C. J. Biol. Chem. 1988; 263: 9831-9839Abstract Full Text PDF PubMed Google Scholar, 9Kong D. Richardson C.C. EMBO J. 1996; 15: 2010-2019Crossref PubMed Scopus (52) Google Scholar, 10Kong D. Nossal N.G. Richardson C.C. J. Biol. Chem. 1997; 272: 8380-8387Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 11Lee J. Chastain P.D., II Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 12Yu M. Masker W. J. Bacteriol. 2001; 183: 1862-1869Crossref PubMed Scopus (13) Google Scholar). However, neither the E. coli SSB protein nor the T4 gene 32 protein can replace gene 2.5 protein in cells infected by T7 phage lacking gene 2.5 (13Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). This specificity for gene 2.5 protein is not surprising as there is little sequence homology between the proteins, and wt gene 2.5 protein differs from the other proteins significantly in a number of biochemical properties. For instance, the T7 protein binds to DNA with a lower affinity than either E. coli SSB protein or T4 gene 32 protein (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar). The oligomeric state of these proteins also differ with wt gene 2.5 protein existing as a stable dimer in solution (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar), whereas E. coli SSB protein forms a tetrameter (14Weiner J.H. Bertsch L.L. Kornberg A. J. Biol. Chem. 1975; 250: 1972-1980Abstract Full Text PDF PubMed Google Scholar), and T4 gene 32 protein is a monomer that forms multimers at high concentrations (15von Hippel P.H. Kowalczykowski S.C. Lonberg N. Newport J.W. Paul L.S. Stormo G.D. Gold L. J. Mol. Biol. 1982; 162: 795-818Crossref PubMed Scopus (76) Google Scholar, 16Carroll R.B. Neet K. Goldthwait D.A. J. Mol. Biol. 1975; 91: 275-291Crossref PubMed Scopus (41) Google Scholar). In addition to interacting with itself, wt gene 2.5 protein also interacts specifically with T7 DNA polymerase and the product of T7 gene 4, a helicase/primase (17Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar).E. coli SSB protein and T4 gene 32 protein feature acidic carboxyl-terminal motifs that are involved in protein-protein interactions (18Curth U. Genschel J. Urbanke C. Greipel J. Nucleic Acids Res. 1996; 24: 2706-2711Crossref PubMed Scopus (118) Google Scholar, 19Genschel J. Curth U. Urbanke C. Biol. Chem. Hoppe-Seyler. 2000; 381: 183-192Crossref PubMed Scopus (99) Google Scholar, 20Burke R.L. Alberts B.M. Hosoda J. J. Biol. Chem. 1980; 255: 11484-11493Abstract Full Text PDF PubMed Google Scholar, 21Reddy M.S. Guhan N. Muniyappa K. J. Biol. Chem. 2001; 276: 45959-45968Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 22Salinas F. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7196-7201Crossref PubMed Scopus (54) Google Scholar). Similarly, the acidic carboxyl terminus of wt gene 2.5 protein is required to mediate interactions with other replication proteins (13Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 23Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar), including those that coordinate leading and lagging strand synthesis by T7 replication proteins on a minicircle template in vitro (11Lee J. Chastain P.D., II Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Because of its critical role in interactions with other replication proteins, mutagenesis studies on gene 2.5 protein to date have focused on the carboxyl terminus (13Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 23Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar). In one study (23Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar), a truncated gene 2.5 protein missing the final 21 amino acids was produced. Expressing this altered gene 2.5 protein in E. coli did not support the growth of a T7 phage deleted in gene 2.5 (23Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar). The truncated gene 2.5 protein itself is a monomer in solution but retains the ability to bind DNA (23Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar). It neither stimulates DNA synthesis by T7 DNA polymerase nor does it interact physically with that protein (23Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar). A second study examined chimeric proteins in which the carboxyl-terminal motif of wt gene 2.5 protein was replaced with the complementary motif of T4 gene 32 protein and E. coli SSB protein (13Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The chimeric proteins could support phage growth, form dimers, and interact with T7 DNA polymerase (13Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). When the carboxyl-terminal motif of T7 wt gene 2.5 protein was used to replace that of E. coli SSB protein and T4 gene 32 protein, the chimeric proteins could not substitute for wt gene 2.5 protein to support the growth of a gene 2.5-deleted phage (13Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These results suggest that although the carboxyl terminus is required for protein-protein interactions, it does not account for the specificity of those interactions (13Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Recently a three-dimensional crystal structure of a carboxyl terminus deleted form of T7 gene 2.5 protein was published (24Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (80) Google Scholar). The protein has a conserved oligosaccharide/oligonucleotide binding fold (25Murzin A.G. EMBO J. 1993; 12: 861-867Crossref PubMed Scopus (774) Google Scholar), similar to that of T4 gene 32 protein (26Shamoo Y. Friedman A.M. Parsons M.R. Konigsberg W.H. Steitz T.A. Nature. 1995; 376: 362-366Crossref PubMed Scopus (221) Google Scholar) and E. coli SSB protein (27Raghunathan S. Ricard C.S. Lohman T.M. Waksman G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6652-6657Crossref PubMed Scopus (191) Google Scholar, 28Raghunathan S. Kozlov A.G. Lohman T.M. Waksman G. Nat. Struct. Biol. 2000; 7: 648-652Crossref PubMed Scopus (361) Google Scholar). The structure suggests models for DNA binding and dimerization (24Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (80) Google Scholar); however, there are no mutagenesis studies to either support or refute these models. In fact, outside of the studies on the carboxyl terminus described above, there is a lack of experimental evidence to define the functional domains of wt gene 2.5 protein. To begin mapping these domains, we developed a screen for lethal mutations in gene 2.5. A similar screen was successfully used to identify lethal mutants of the T7 helicase/primase (29Rosenberg A.H. Griffin K. Washington M.T. Patel S.S. Studier F.W. J. Biol. Chem. 1996; 271: 26819-26824Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Presumably, mutations that are lethal will occur in regions critical to wt gene 2.5 protein functions or proper folding. In the present study we characterize three of the altered proteins biochemically, and we show that they define the interface for dimer formation, demonstrating that dimerization is an essential property of gene 2.5 protein. E. coli XL1-Red (endA1 gyrA96 thi1 hsdR17supE44 relA1 lac mutD5 mutSmutT Tn10 (Tetr)) (Stratagene) was used to generate a library of randomly mutated plasmids. E. coli HMS262 (F− hsdR pro leu − lac − thi − supE tonA − trxA−) and E. coli HMS 89 (xth1 thi argE mtl xyl str-R ara his galK lacY proA leu thr tsx supE) were used as hosts for phage experiments.E. coli BL21 (DE3) (F− ompT hsdS B(rB−mB−) gal dcm (DE3)) (Novagen) was used to express wild-type gene 2.5 and mutant gene 2.5. Construction of the T7 deletion phage (T7Δ2.5) was described previously (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (59) Google Scholar). T7Δ2.5 phage used in the in vivo DNA synthesis experiments was provided by Jaya Kumar (Harvard Medical School). The plasmids encoding gene 2.5, pETGP2.5 and pETGP2.5-PPS were provided by James Stattel (Harvard Medical School). The parent vector of pETGP2.5-PPS, pET19bPPS, which encodes a tag of 10 histidine residues and a rhinovirus C protease (PreScission protease, Amersham Biosciences) cleavage site located upstream of the start codon, was kindly provided by Tapan Biswas (Harvard Medical School). The following oligonucleotides were purchased from Oligos Etc.: T72.5NdeI, 5′-CGTAGGATCCATATGGCTAAGAAGATTTTCACCTC-3′; T72.5BamHI, 5′-CGTAGGATCCACTTAGAAGTCTCCGTC-3′; and Oligo 70, 5′-GACCATATCCTCCACCCTCCCCAATATTGACCATCAACCCTTCACCTCACTTCACTCCACTATACCACTC-3. The following oligonucleotides were purchased from Integrated DNA Technologies: T7 promoter, 5′-TAATACGACTCACTATAGGGG-3′; pET17up, 5′-CTTTAAGAAGGAGATATACATATG-3′; T7 terminator, 5′-GCTAGTTATTGCTCAGCGG-3′; and DS17b, 5′-GCTTCCTTTCGGGCTTTG-3′. The oligonucleotide BCMP206, 5′-TAACGCCAGGGTTTTCCCAGTCACG-3′, was synthesized by the Biopolymer Laboratory, Harvard Medical School. M13 (mGP1-2) DNA and T7 DNA polymerase lacking exonuclease activity (30Tabor S. Richardson C.C. J. Biol. Chem. 1989; 264: 6447-6458Abstract Full Text PDF PubMed Google Scholar) were kindly provided by Stan Tabor (Harvard Medical School). Wild-type and altered gene 2.5 proteins were purified as described below. Gene 2.5 protein-Δ26C was provided by Eric Toth (Harvard Medical School). His-gene 2.5 protein-Δ26C was provided by James Stattel (Harvard Medical School). T7 DNA polymerase was provided by Don Johnson and Joon-Soo Lee (Harvard Medical School). A library of randomly mutated plasmids was created using the mutator E. coli strain XL1-Red (Stratagene). The plasmid pETGP2.5 was transformed into XL1-Red, and transformants were plated on LB plates supplemented with 60 μg/ml ampicillin and incubated overnight at 37 °C. The next day, 2 ml of LB were added to plates to facilitate the scraping of the colonies. Ampicillin was added to a concentration of 60 μg/ml, and the culture of pooled colonies were grown overnight at 37 °C. The next day plasmid DNA was prepared from the bacteria using an RPM kit (Qbiogene). Selection of lethal mutations in gene 2.5 was based on the complementation assay described previously (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (59) Google Scholar). When gene 2.5 is expressed on a plasmid, the phage T7Δ2.5 can grow in E. coli HMS262. The screen was performed in a manner similar to that used to uncover lethal mutants of bacteriophage T7 gene 4 (29Rosenberg A.H. Griffin K. Washington M.T. Patel S.S. Studier F.W. J. Biol. Chem. 1996; 271: 26819-26824Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) with alterations noted below. Randomly mutated plasmids generated from pETGP2.5 were introduced intoE. coli HMS262 by electroporation using an E. coli Pulser Transformation Apparatus (Bio-Rad) in 19 separate experiments. Electrocompetent E. coli HMS262 cells were prepared according to the manufacturer's recommendation (Bio-Rad). In each experiment, 1 ng of DNA was mixed with 40 μl of electrocompetent cells and incubated on ice for 5 min. The mixtures were transferred to 0.1-cm cuvettes (Bio-Rad). Cuvettes were pulsed at 1.80 kV. One ml of SOC (2% bactotryptone, 0.5% yeast extract, 10 mm NaCl, 2.5 mm KCl, 10 mm MgSO4, 20 mm glucose) was added immediately after pulsing, and the mixture was then transferred to a 15-ml polystyrene tube. Cells were allowed to recover by shaking for 1 h at 37 °C. One hundred fifty μl of cells were plated on LB plates containing 60 μg/ml ampicillin, which were overlaid with 2.5 ml of top agar (1% tryptone, 0.5% yeast, 0.5% NaCl, 0.7% agar (pH 7.0)) containing 60 μg/ml ampicillin either alone or with 107 plaque-forming units of T7Δ2.5 phage. Plates were incubated at 37 °C overnight. The next morning, colonies that formed on the LB plates with ampicillin were counted to determine the efficiency of electroporation. Colonies that formed on the plates overlaid with T7Δ2.5 phage were counted, then streaked on LB plates with 60 μg/ml ampicillin, and cross-streaked with T7Δ2.5 phage to confirm that the cells could not support the replication of the gene 2.5 deleted phage. Approximately 0.6% of the colonies screened could not support the growth of T7Δ2.5 phage. After streaking, a collection of 291 cultures of transformants that are unable to support the growth of T7Δ2.5 phage were frozen as glycerol stocks. Plasmid DNA was prepared from 5-ml cultures of 216 independent transformants. Each plasmid was analyzed by restriction digests with NdeI and BamHI (New England Biolabs) to ensure that a 699-bp fragment was released. This analysis eliminated 14 plasmids from further consideration. The remaining 202 plasmids were sequenced by the Dana-Farber/Harvard Cancer Center High-Throughput DNA Sequencing Facility using the sequencing primers pET17up and DS17b. Readable sequence was obtained for 190 plasmids. DNA synthesis was measured by a method modified from Richardson and co-workers (31Saito H. Richardson C.C. J. Virol. 1981; 37: 343-351Crossref PubMed Google Scholar, 32Kumar J.K. Kremsdorf R. Tabor S. Richardson C.C. J. Biol. Chem. 2001; 276: 46151-46159Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). A culture of Davis minimal media supplemented with 60 μg/ml ampicillin was inoculated with E. coli HMS262 transformed with pETGP2.5, pETGP2.5-P22L, pETGP2.5-F31S, or pETGP2.5-G36S and grown at 30 °C in a gyratory shaker. Cells were grown to a density of 3 × 108 cells per ml and then infected with T7Δ2.5 phage at a multiplicity of infection of 7. At 5-min intervals post-infection, 200-μl samples were removed, and [3H]thymidine (50 μCi/ml) was added. Reactions were incubated at 30 °C for 90 s and then terminated by adding 40 μl of an ice-cold solution of 50 mm Tris-HCl (pH 7.5), 2 mm EDTA, 2% SDS. Sixty μl of the terminated reactions were spotted onto DE81 filters. Filters were washed 3 times in 0.3 m ammonium formate, 2 times in ethanol, and then air-dried. [3H]Thymidine incorporation into DNA was then determined by liquid scintillation counting. Wild-type and altered gene 2.5 protein were purified by a procedure developed by Stattel and Richardson. 2J. Stattel and C. C. Richardson, unpublished data. The plasmids pET2.5, pET2.5-P22L, pET2.5-F31S, and pET2.5-G36S were transformed into E. coli BL21(DE3) (Novagen). One- (pET2.5-P22L and pET2.5-F31S) or 8-liter cultures (pET2.5, pET2.5-G36S) were grown in LB with 60 μg/ml ampicillin to an OD of 1.0. Cells were induced for 4 h after adding isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 1 mm. Cells were then collected by centrifugation and resuspended in 20 ml/liter of culture lysis buffer (50 mm Tris-Cl (pH 7.5), 0.1 mm EDTA, 10% sucrose), frozen in dry ice, and stored at −70 °C. Lysozyme (Sigma) was added to thawed cells (final concentrated 1 mg/ml) and stirred in the cold for 1 h. Lysed cells were warmed to 20 °C in a 37 °C bath, then chilled on ice, and centrifuged at 4 °C for 45 min at 100,000 × g. Polyethyleneimine (pH 7.5) was added to the supernatant (final concentration, 0.1%), and the mixture was stirred at 4 °C for 1 h. The mixture was centrifuged at 4 °C for 15 min at 21,000 × g. The resulting pellet was suspended in 75 ml of Buffer A (50 mm Tris-Cl (pH 7.5), 0.1 EDTA, 1 mm dithiothreitol (DTT), 10% glycerol) containing 1 m NaCl, stirred for 1 h at 4 °C, and then centrifuged at 21,000 × g for 15 min at 4 °C. The supernatant was collected and then diluted with Buffer A to a final volume of 150 ml. To precipitate the proteins, (NH4)2SO4 was added to 80% saturation, and the solution was stirred for 1 h at 4 °C and then centrifuged at 21,000 × g for 15 min. The pellet was suspended in 60 ml of Buffer A and filtered through a 0.22-μm syringe filter. The sample was loaded onto a POROS HQ column (PE Biosystems) and gene 2.5 protein eluted in a 50 mm to 1m NaCl gradient. Fractions containing gene 2.5 protein were pooled, and the protein was precipitated by adding (NH4)2SO4 to 60% saturation. The solution was centrifuged at 21,000 × g for 15 min. The resulting pellet was resuspended in Buffer G (50 mmKPO4 (pH 7.0), 150 mm NaCl, 0.1 mmEDTA, 0.1 mm DTT, and 10% glycerol) to a concentration of no more than 5 mg/ml. The sample was loaded onto a Superose 12 column (Amersham Biosciences). Fractions that contained gene 2.5 protein were pooled, dialyzed against Buffer S (50 mm Tris-Cl (pH 7.5), 0.1 mm EDTA, 1 mm DTT, 50% glycerol), and then stored at −20 °C. Purified wt gene 2.5 protein, gene 2.5 protein-P22L, and gene 2.5 protein-F31S were over 99% pure as determined by denaturing polyacrylamide gel electrophoresis followed by Coomassie Blue staining and were free of contaminating DNA-dependent nuclease activity (data not shown). Protein concentrations were calculated from UV spectrophotometer readings at 280 mm, using the calculated extinction coefficients at 280 nm (33Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5060) Google Scholar) of 2.58 × 104m−1 cm−1. This procedure consistently yielded only small amounts of gene 2.5 protein-G36S, and the preparations were contaminated with a DNA nuclease. For this reason gene 2.5 protein-G36S was expressed and purified as a 10-histidine fusion protein as described below. Separate 1-liter cultures of BL21(DE3) cells transformed with pET19b2.5PPS, pET19b2.5PPS-P22L, pET19b2.5PPS-F31S, and pET19b2.5PPS-G36S were grown, induced, and harvested as described above. Pellets were resuspended in 20 ml of Buffer B (50 mmTris-Cl (pH 7.5), 500 mm NaCl) containing 70 mmimidazole, then frozen in dry ice, and stored at −70 °C. Lysozyme (Sigma) was added to thawed cells (final concentration 1 mg/ml), and the suspension was stirred at 4 °C for 2 h. One hundred twenty five units of Benzonase nuclease (Novagen) was added to lysates that were then rapidly warmed to ∼20 °C in a 37 °C bath, chilled on ice, and centrifuged at 4 °C for 1 h at 8,000 ×g. Supernatants were loaded onto a 5-ml column packed with nickel-NTA-agarose (Qiagen). The column was washed with 10 column volumes of Buffer B containing 70 mm imidazole and proteins eluted in 2 column volumes of Buffer B containing 500 mmimidazole. Histidine-tagged gene 2.5 protein (His-gene 2.5 protein), His-gene 2.5 protein-P22L, His-gene 2.5 protein-F31S, and His-gene 2.5 protein-G36S were dialyzed against Buffer S, and stored at −20 °C. An aliquot of His-gene 2.5 protein-G36S was then processed to remove the amino-terminal tag. To cleave the histidine tag, 50 μg of PreScission protease was added to the eluted fraction, and the entire protein solution was dialyzed for18 h against Buffer C (50 mm Tris-Cl (pH 8.0), 225 mm NaCl, 0.1 mm EDTA, 2 mm DTT) using 10-kDa cut-off dialysis membrane (Pierce). The dialyzed protein solution was passed through a 1-ml GSTrap column (Amersham Biosciences) at a rate of 0.5 ml/min to remove the PreScission protease. Proteins were then re-applied to a 5-ml Ni-NTA column to ensure removal of any protein that still contained the histidine tag. Purified proteins were dialyzed into Buffer S and stored at −20 °C. Proteins prepared in this manner were determined to be over 95% pure and free of contaminating nuclease activity. Gel filtration analysis was performed as described previously (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar). Briefly, in three independent experiments 50 μg of wt gene 2.5 protein, gene 2.5 protein-P22L, gene 2.5 protein-F31S, and gene 2.5 protein-G36S diluted in Buffer S (final concentration 4 μm) were applied to a Superdex 75 column (Amersham Biosciences) at a flow rate of 0.50 ml/min. The elution of each protein was monitored by absorbance at 280 nm. Chromatography was carried out at 4 °C in Buffer G (50 mm KPO4 (pH 7.0), 150 mm NaCl, 0.1 mm EDTA, 0.1 mm DTT, and 10% glycerol). The running buffer for high salt experiments was 50 mmKPO4 (pH 7.0), 250 mm NaCl, 0.1 mmEDTA, 0.1 mm DTT, and 10% glycerol. The peak elution volume (v e) was taken to be the average of the volumes at which each protein eluted in three experiments. The void volume (v 0) and total volume (v t) were determined by independently applying blue dextran and xylene cyanol, respectively. The fractional retention (K av) was calculated using the formulaK av = (v e −v 0)/(v t −v 0), where v e is the peak elution volume. A standard curve of K av versus log M r was generated by applying both high and low molecular weight protein standards (AmershamBiosciences) to the column under the conditions described above. Standard curves were generated at both salt concentrations examined in this study. The oligodeoxynucleotide 70 was end-labeled using T4 polynucleotide kinase (New England Biolabs) and [γ-33P]ATP and then purified using micro BioSpin 6 chromatography columns (Bio-Rad). The 15-μl reactions included 3 nm33P-labeled 70-mer oligonucleotide, 15 mm MgCl2, 5 mm DTT, 50 mm KCl, 10% glycerol, 0.01% bromphenol blue, and various concentrations (from 0 to 10 μm) of either wt gene 2.5 protein, gene 2.5 protein-P22L, gene 2.5 protein-F31S, or gene 2.5 protein-G36S diluted in a buffer of 20 mm Tris (pH 7.5), 10 mm β-mercaptoethanol, and 500 μg/ml bovine serum albumin. Reactions were immediately put on ice and then loaded onto a 10% TBE Ready Gel (Bio-Rad) running in 0.5× Tris/glycine buffer (12.5 mm Tris base, 95 mm glycine, and 0.5 mm EDTA). Gels were run at 80 V for 2 h at 4 °C and then dried and exposed to a Fujix PhosphorImager plate for quantitation using ImageQuant software. Dissociation constants were calculated from the average of three experiments using the Langmuir isotherm formula. In the experiments where the salt concentration was varied, KCl was replaced by NaCl at a variety of concentrations (0, 50, 100, 150, 200, 250, 300, or 400 mm). In these experiments gene 2.5 protein concentration was 1.3 μm. The ability of wt gene 2.5 protein to facilitate the annealing of homologous strands of DNA was assessed using an in vitro annealing assay developed by Tabor and Richardson. 3S. Tabor and C. C. Richardson, unpublished data. The assay measures the annealing of a radiolabeled ssDNA fragment of M13 DNA to unlabeled circular M13 ssDNA. The labeled fragment was generated in a 60.5-μl reaction by annealing 60 pmol of the oligonucleotide BCMP206 to 8 pmol of M13 (mp1-2) in a buffer containing 25 mmTris-Cl (pH 7.5), and 50 mm NaCl. The annealed primer was partially extended by T7 DNA polymerase-Δ28 in a 77.75-μl reaction containing 10 mm MgCl2, 3.9 mm DTT, 0.13 mg/ml bovine serum albumin, 2.5 μCi [α−32P]dGTP, and a limiting (8 μm each) quantity of dATP, dCTP, dGTP, and dTTP. After 10 min at room temperature, the reaction was supplemented with 80 μmeach of dATP, dCTP, dGTP, and dTTP, and DNA synthesis was completed in 15 min at room temperature. Reactions were then incubated for 10 min at 70 °C to inactivate the polymerase. Next, E. coli SSB protein was added, and the DNA was digested with Acc65-1 (New England Biolabs) for 2 h at 37 °C. Reactions were extracted with phenol/chloroform/isoamyl alcohol (50:49:1), and DNA was purified using microspin S-400 columns (Amersham Biosciences). ssDNA fragments were generated by adding NaOH to a final concentration of 100 mmand incubating at room temperature for 5 min. HCl and Tris-Cl (pH 7.5) were each added to a final concentration of 100 mm, and DNA fragments were separated on a 1.4% agarose gel. After electrophoresis the 310-bp band was cut from the gel, and DNA was isolated using a QIAquick gel extraction kit (Qiagen). DNA annealing was assayed in 20-μl reactions containing 4 nm32P-labeled ssDNA fragment, 20 μm M13 mGP1–2 ssDNA, 20 mm Tris-Cl (pH 7.5), 1 mm DTT, 10 mm MgCl2, 50 mm NaCl, and 0–30 μm wt gene 2.5 protein or altered gene 2.5 proteins. Reactions were incubated at 30 °C for 8 min and then analyzed on a 0.8% agarose gel at 75 V for 1 h at room temperature, then dried, and exposed to a Fujix PhosphorImager plate. Time course experiments were carried out under the same conditions except all reactions contained a constant amount of gene 2.5 protein (gene 2.5 protein, 10 μm; gene 2.5 protein-P22L, 10 μm; gene 2.5 protein-F31S, 10 μm; gene 2.5 protein-G36S, 30 μm). Reactions were stopped by adding SDS to a final concentration of 0.5% and then immediately put on ice. The interaction between gene 2.5 protein and T7 DNA polymerase was assayed by surface plasmon resonance (SPR) using the BIAcore 3000 system. Histidine-tagged gene 2.5 protein, gene 2.5 protein-P22L, gene 2.5 protein-F31S, g
• Rezende, L. F., Hollis, T., Ellenberger, T., & Richardson, C. C. (2002). Essential amino acid residues in the single-stranded DNA-binding protein of bacteriophage T7. Identification of the dimer interface. The Journal of biological chemistry, 277(52), 50643-53.
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  Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein. T7 phage with gene 2.5 deleted can grow only on Escherichia coli cells that express gene 2.5 from a plasmid. This complementation assay was used to screen for lethal mutations in gene 2.5. By screening a library of randomly mutated plasmids encoding gene 2.5, we identified 20 different single amino acid alterations in gene 2.5 protein that are lethal in vivo. The location of these essential residues within the three-dimensional structure of gene 2.5 protein assists in the identification of motifs in the protein. In this study we show that a subset of these alterations defines the dimer interface of gene 2.5 protein predicted by the crystal structure. Recombinantly expressed and purified gene 2.5 protein-P22L, gene 2.5 protein-F31S, and gene 2.5 protein-G36S do not form dimers at salt concentrations where the wild-type gene 2.5 protein exists as a dimer. The basis of the lethality of these mutations in vivo is not known because altered proteins retain the ability to bind single-stranded DNA, anneal complementary strands of DNA, and interact with T7 DNA polymerase.
• Rezende, L. F., Kew, Y., & Prasad, V. R. (2001). Forward mutation rate of human immunodeficiency virus type 1 reverse transcriptase in vitro: Effect of increased processivity on overall fidelity and error specificity,". Journal of Biomedical Science, 8(2), 197-205.
• Rezende, L. F., Kew, Y., & Prasad, V. R. (2001). The Effect of Increased Processivity on Overall Fidelity of Human Immunodeficiency Virus Type 1 Reverse Transcriptase. Journal of Biomedical Science. doi:10.1159/000054033
• Drosopoulos, W. C., Rezende, L. F., Wainberg, M. A., & Prasad, V. R. (1998). Virtues of being faithful: Can we limit genetic variation in human immunodeficiency virus?. Journal of Molecular Medicine, 76(9), 604-612.
• Drosopoulos, W. C., Rezende, L. F., Wainberg, M. A., & Prasad, V. R. (1998). Virtues of being faithful: can we limit the genetic variation in human immunodeficiency virus?. Journal of Molecular Medicine. doi:10.1007/s001090050257
• Rezende, L. F., Curr, K., Ueno, T., Mitsuya, H., & Prasad, V. R. (1998). The Impact of Multidideoxynucleoside Resistance-Conferring Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Polymerase Fidelity and Error Specificity. Journal of Virology. doi:10.1128/jvi.72.4.2890-2895.1998
• Rezende, L. F., Drosopoulos, W. C., & Prasad, V. R. (1998). The influence of 3TC-resistance mutation M184I on the fidelity and error specificity of human immunodeficiency virus type 1 reverse transcriptase. Nucleic Acids Research, 26(12), 3066-3072.
• Rezende, L. F., Ueno, T., Mitsuya, H., & Prasad, V. R. (1998). The impact of multi-dideoxynucleoside resistance-conferring mutations in human immunodeficiency virus type 1 on fidelity and error specificity of reverse transcriptase. Journal of Virology, 72(4), 2890-2895.
• Hsu, M., Inouye, P., Rezende, L. F., Richard, N., Li, Z., Prasad, V., & Wainberg, M. A. (1997). Higher fidelity of RNA-dependent DNA mispair extension by M184V drug-resistant than wild-type reverse transcriptase of human immunodeficiency virus type 1. Nucleic Acids Research, 25(22), 4532-4536.

Proceedings Publications

• Yi, R. H., Welcsch, P., Rezende, L. F., Dearfield, C. T., Owens, K. N., & Friedman, S. J. (2020, December). Abstract P1-15-07: Effectiveness of an online educational resource in increasing lay users' understanding of information in media reports on breast cancer research. In San Antonio Breast Cancer Symposium.
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  Abstract Rationale: Breast cancer diagnosis at a young age is associated with higher risk of recurrence, second malignancy, mortality, morbidity, and impact on quality of life. Young breast cancer survivors face more life-years after treatment, with associated financial, emotional, and physical burdens. Long-term effects of treatment may include early menopause, fertility impairment, neuropathy, cardiovascular disease, lowered bone density, and risk of second malignancies. Young women with or at-risk for breast cancer often seek health information through mainstream print and digital media. However, they often find it does not address their unique concerns, is difficult to interpret, or even misleading. Media reports of breast cancer research are often unreliable, misleading, or confusing regarding which information is clinically relevant. Common flaws include exaggerating prevalence, ignoring potential side effects of treatment, and failing to discuss all treatment options. Young women with or at-risk for breast cancer, need accurate, clearly presented information based on sound evidence to help them make informed decisions about their specific health needs. To help women better understand media coverage about new research, Facing Our Risk of Cancer Empowered (FORCE) developed the CDC-funded XRAYS (eXamining Relevance of Articles to Young Survivors) program. XRAYS is an online resource that provides brief articles summarizing recent research relevant to young women with or at-risk for breast cancer. XRAYS articles rate the quality and relevance of research, the quality of media reporting, and suggest questions that may be useful to address with health care providers. One critical aim of XRAYS is to improve users’ understanding of the limitations of research methods and of media reporting. FORCE charged an independent evaluator with conducting an assessment of XRAYS’ effects on users’ understanding of methodological and reporting issues in media articles about research related to breast cancer. Objective: The objective of this study was to assess whether XRAYS improves readers’ understanding of limitations of study methods and media reporting more than reading media reports alone. Methods: To assess XRAYS’ impact on users’ understanding of limitations in research methods and reporting quality, an independent evaluator conducted a study with 36 volunteer participants who were attending a FORCE conference. Participants were randomly assigned to treatment or control groups. The treatment group read a media report and the corresponding XRAYS article. The control group read only the media report. After reading the materials, participants answered paper and pencil multiple choice questions about methodological limitations of the research discussed in the media report, and about limitations of the reporting itself. Results: Results are summarized in the table. GroupNMean Score (s.d.)t-score (d.f.)PComparison Group1742.85 (19.85)-3.08 (34)
• Yi, R. P., Rezende, L. F., Dearfield, C., Welcsh, P., & Friedman, S. (2019, December). Abstract P6-14-08: Effects of online resource to support laypersons' understanding of media reports on breast cancer research. In San Antonio Breast Cancer Symposium.
• Holman, L. L., Chen, A., Zhao, D., Dockery, L., Rezende, L. F., & Friedman, S. (2017). Decision-making surrounding genetic testing among women with ovarian carcinoma. In Society of Gynecologic Oncology.
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  Objective: While current guidelines recommend BRCA testing for all women diagnosed with ovarian cancer (OC), uptake of this testing is variable. We aimed to investigate factors surrounding decision making regarding BRCA testing among women with OC.

Presentations

• Marsteller, P., Beaulieu, E., Bill, B., Flaherty, D., Gass, S., Rezende, L. F., & Zwich, M. (2022, July). Applying Universal Design for Learning Principles to Case Studies. BIOME Annual Conference. Online: BIOME.
• Rezende, L. F., & Romanoski, S. (2022, May). Changing Patient Advocates Attitudes and Practice: Lessons from Escape to Thrive. Escape to Thrive. Tucson, Arizona: BagIt.
• Elfring, L. K., Rezende, L. F., Lattimore, K. L., & Hester, S. D. (2021). An Instructional-Teams Project for supporting instructional reform. 2021 ASCN Transforming Institutions Conference.
• Rezende, L. F. (2019, February). Communicating Complex Science to the Public: Lessons from the XRAYS Program. NIH CSER Consortium Engagement Work Group Meeting. Webinar: NIH CSER Consortium.
• Rezende, L. F. (2016, November). Experiences Talking to Family Members About Inherited Mutations That Increase Cancer Risk: Results from the ABOUT Network Family Communication Survey. Michigan Department of Health and Human Services Cancer Genomics Webinar Series. Webinar: Michigan Department of Health and Human Services.
• Rezende, L. F. (2015, March). Finding the gaps between national guidelines and patient decisions in the hereditary cancer community. NIH Collaboratory Grand Rounds. Webinar: NIH Collabatory.
• Rezende, L. F., & Schlager, L. (2014, November). BRCA Positive: Now What?”. International Society of Nurses in Genetics World ConferenceInternational Society of Nurses in Genetics.

Poster Presentations

• Pugh Yi, R. H., Welcsh, P., Rezende, L., Dearfiled, C., Owens, K., & Friedman, S. J. (2019, December). Effectiveness of an online educational resource in increasing lay users' understanding of information in media reports on breast cancer research. San Antonio Breast Cancer Symposium.
• Pugh Yi, R. H., Welscsh, P., Rezende, L., Dearfield, C., Owens, K., & Friedman, S. J. (2019, December). Effects of an online educational resource on lay audience understanding of limitations of quality in media reports and research methods. San Antonio Breast Cancer Symposium. San Antonio, TX.
• Pugh Yi, R. H., Rezende, L. F., Dearfield, C. T., Welcsh, P. L., & Friedman, S. J. (2018, December). Effects of Online Resource to Support Laypersons’ Understanding of Media Reports on Breast Cancer Research. San Antonio Breast Cancer Symposium. San Antonio.
• Lisa, S., Rezende, L. F., Friedman, S., Cohen, S., & Rose, D. (2017, November 3). Peer Navigation Benefits People Affected by Hereditary Cancers. International Society of Nurses in Genetics World Congress. Reston, VA: International Society of Nurses in Genetics.
• Schlager, L., Rezende, L. F., Friedman, S., Huynh, J., & Pugh Yi, R. (2017, November 3). XRAYS: Unconfuse the News for Young Breast Cancer Survivors and Those at High Risk of Breast Cancer. International Society of Nurses in Genetics World Congress. Reston, VA: International Society of Nurses in Genetics.