Andrew L Paek
- Assistant Professor, Molecular and Cellular Biology
- Ph.D. Molecular and Cellular Biology
- University of Arizona, Tucson, Arizona, United States
- Formation of Dicentric and Acentric Chromosomes, by a Template Switch Mechanism, in Budding Yeast
- B.S. Applied Mathematics
- University of Texas, Austin, Texas, United States
- B.S. Microbiology
- University of Texas, Austin, Texas, United States
- Postdoctoral Fellow, Harvard Medical School, Boston, Massachusetts (2011 - 2016)
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- Paek, A. L., Liu, J. C., Loewer, A., Forrester, W. C., & Lahav, G. (2016). Cell-to-Cell Variation in p53 Dynamics Leads to Fractional Killing. Cell, 165(3), 631-42.More infoMany chemotherapeutic drugs kill only a fraction of cancer cells, limiting their efficacy. We used live-cell imaging to investigate the role of p53 dynamics in fractional killing of colon cancer cells in response to chemotherapy. We found that both surviving and dying cells reach similar levels of p53, indicating that cell death is not determined by a fixed p53 threshold. Instead, a cell's probability of death depends on the time and levels of p53. Cells must reach a threshold level of p53 to execute apoptosis, and this threshold increases with time. The increase in p53 apoptotic threshold is due to drug-dependent induction of anti-apoptotic genes, predominantly in the inhibitors of apoptosis (IAP) family. Our study underlines the importance of measuring the dynamics of key players in response to chemotherapy to determine mechanisms of resistance and optimize the timing of combination therapy.
- U'Ren, J. M., Wisecaver, J. H., Paek, A. L., Dunn, B. L., & Hurwitz, B. L. (2015). Draft Genome Sequence of the Ale-Fermenting Saccharomyces cerevisiae Strain GSY2239. Genome announcements, 3(4).More infoSaccharomyces cerevisiae strain GSY2239 is derived from an industrial yeast strain used to ferment ale-style beer. We present here the 11.5-Mb draft genome sequence for this organism.
- Carr, A. M., Paek, A. L., & Weinert, T. (2011). DNA replication: failures and inverted fusions. Seminars in cell & developmental biology, 22(8), 866-74.More infoDNA replication normally follows the rules passed down from Watson and Crick: the chromosome duplicates as dictated by its antiparallel strands, base-pairing and leading and lagging strand differences. Real-life replication is more complicated, fraught with perils posed by chromosome damage for one, and by transcription of genes and by other perils that disrupt progress of the DNA replication machinery. Understanding the replication fork, including DNA structures, associated replisome and its regulators, is key to understanding how cells overcome perils and minimize error. Replication fork error leads to genome rearrangements and, potentially, cell death. Interest in the replication fork and its errors has recently gained added interest by the results of deep sequencing studies of human genomes. Several pathologies are associated with sometimes-bizarre genome rearrangements suggestive of elaborate replication fork failures. To try and understand the links between the replication fork, its failure and genome rearrangements, we discuss here phases of fork behavior (stall, collapse, restart and fork failures leading to rearrangements) and analyze two examples of instability from our own studies; one in fission yeast and the other in budding yeast.
- Kaochar, S., Paek, A. L., & Weinert, T. (2010). Genetics. Replication error amplified. Science (New York, N.Y.), 329(5994), 911-3.
- Paek, A. L., & Weinert, T. (2010). Choreography of the 9-1-1 checkpoint complex: DDK puts a check on the checkpoints. Molecular cell, 40(4), 505-6.More infoCheckpoint proteins respond to DNA damage by halting the cell cycle until the damage is repaired. In this issue of Molecular Cell, Furuya et al. (2010) provide evidence that checkpoint proteins need to be removed from sites of damage in order to properly repair it.
- Paek, A. L., Jones, H., Kaochar, S., & Weinert, T. (2010). The role of replication bypass pathways in dicentric chromosome formation in budding yeast. Genetics, 186(4), 1161-73.More infoGross chromosomal rearrangements (GCRs) are large scale changes to chromosome structure and can lead to human disease. We previously showed in Saccharomyces cerevisiae that nearby inverted repeat sequences (∼20-200 bp of homology, separated by ∼1-5 kb) frequently fuse to form unstable dicentric and acentric chromosomes. Here we analyzed inverted repeat fusion in mutants of three sets of genes. First, we show that genes in the error-free postreplication repair (PRR) pathway prevent fusion of inverted repeats, while genes in the translesion branch have no detectable role. Second, we found that siz1 mutants, which are defective for Srs2 recruitment to replication forks, and srs2 mutants had opposite effects on instability. This may reflect separate roles for Srs2 in different phases of the cell cycle. Third, we provide evidence for a faulty template switch model by studying mutants of DNA polymerases; defects in DNA pol delta (lagging strand polymerase) and Mgs1 (a pol delta interacting protein) lead to a defect in fusion events as well as allelic recombination. Pol delta and Mgs1 may collaborate either in strand annealing and/or DNA replication involved in fusion and allelic recombination events. Fourth, by studying genes implicated in suppression of GCRs in other studies, we found that inverted repeat fusion has a profile of genetic regulation distinct from these other major forms of GCR formation.
- Paek, A. L., Kaochar, S., Jones, H., Elezaby, A., Shanks, L., & Weinert, T. (2009). Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast. Genes & development, 23(24), 2861-75.More infoLarge-scale changes (gross chromosomal rearrangements [GCRs]) are common in genomes, and are often associated with pathological disorders. We report here that a specific pair of nearby inverted repeats in budding yeast fuse to form a dicentric chromosome intermediate, which then rearranges to form a translocation and other GCRs. We next show that fusion of nearby inverted repeats is general; we found that many nearby inverted repeats that are present in the yeast genome also fuse, as does a pair of synthetically constructed inverted repeats. Fusion occurs between inverted repeats that are separated by several kilobases of DNA and share >20 base pairs of homology. Finally, we show that fusion of inverted repeats, surprisingly, does not require genes involved in double-strand break (DSB) repair or genes involved in other repeat recombination events. We therefore propose that fusion may occur by a DSB-independent, DNA replication-based mechanism (which we term "faulty template switching"). Fusion of nearby inverted repeats to form dicentrics may be a major cause of instability in yeast and in other organisms.
- Weinert, T., Kaochar, S., Jones, H., Paek, A., & Clark, A. J. (2009). The replication fork's five degrees of freedom, their failure and genome rearrangements. Current opinion in cell biology, 21(6), 778-84.More infoGenome rearrangements are important in pathology and evolution. The thesis of this review is that the genome is in peril when replication forks stall, and stalled forks are normally rescued by error-free mechanisms. Failure of error-free mechanisms results in large-scale chromosome changes called gross chromosomal rearrangements, GCRs, by the aficionados. In this review we discuss five error-free mechanisms a replication fork may use to overcome blockage, mechanisms that are still poorly understood. We then speculate on how genome rearrangements may occur when such mechanisms fail. Replication fork recovery failure may be an important feature of the oncogenic process. (Feedback to the authors on topics discussed herein is welcome.).
- Bolusani, S., Ma, C. H., Paek, A., Konieczka, J. H., Jayaram, M., & Voziyanov, Y. (2006). Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites. Nucleic acids research, 34(18), 5259-69.More infoAs a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites.
- Ghosh, S. K., Hajra, S., Paek, A., & Jayaram, M. (2006). Mechanisms for chromosome and plasmid segregation. Annual review of biochemistry, 75, 211-41.More infoThe fundamental problems in duplicating and transmitting genetic information posed by the geometric and topological features of DNA, combined with its large size, are qualitatively similar for prokaryotic and eukaryotic chromosomes. The evolutionary solutions to these problems reveal common themes. However, depending on differences in their organization, ploidy, and copy number, chromosomes and plasmids display distinct segregation strategies as well. In bacteria, chromosome duplication, likely mediated by a stationary replication factory, is accompanied by rapid, directed migration of the daughter duplexes with assistance from DNA-compacting and perhaps translocating proteins. The segregation of unit-copy or low-copy bacterial plasmids is also regulated spatially and temporally by their respective partitioning systems. Eukaryotic chromosomes utilize variations of a basic pairing and unpairing mechanism for faithful segregation during mitosis and meiosis. Rather surprisingly, the yeast plasmid 2-micron circle also resorts to a similar scheme for equal partitioning during mitosis.
- Konieczka, J. H., Paek, A., Jayaram, M., & Voziyanov, Y. (2004). Recombination of hybrid target sites by binary combinations of Flp variants: mutations that foster interprotomer collaboration and enlarge substrate tolerance. Journal of molecular biology, 339(2), 365-78.More infoStrategies of directed evolution and combinatorial mutagenesis applied to the Flp site-specific recombinase have yielded recombination systems that utilize bi-specific hybrid target sites. A hybrid site is assembled from two half-sites, each harboring a distinct binding specificity. Satisfying the two specificities by a binary combination of Flp variants, while necessary, may not be sufficient to elicit recombination. We have identified amino acid substitutions that foster interprotomer collaboration between partner Flp variants to potentiate strand exchange in hybrid sites. One such substitution, A35T, acts specifically in cis with one of the two partners of a variant pair, Flp(K82M) and Flp(A35T, R281V). The same A35T mutation is also present within a group of mutations that rescue a Flp variant, Flp(Y60S), that is defective in establishing monomer-monomer interactions on the native Flp target site. Strikingly, these mutations are localized to peptide regions involved in interdomain and interprotomer interactions within the recombination complex. The same group of mutations, when transferred to the context of wild-type Flp, can relax its specificity to include non-native target sites. The hybrid Flp systems described here mimic the naturally occurring XerC/XerD recombination system that utilizes two recombinases with distinct DNA binding specificities. The ability to overcome the constraints of binding site symmetry in Flp recombination has important implications in the targeted manipulations of genomes.