The principal goal of the proposed research is to study the biochemistry and genetics of replication of yeast chromosomal DNA. In particular, the work will focus on two projects: (1) elucidation of the mechanisms that contribute to the fidelity of DNA replication, and (2) determination of the in vivo replication fork. The yeast DNA polymerase I-exonuclease complex will be purified and the fidelity of DNA replication determined in in vitro assays. Using antibodies directed against the exonuclease subunit or a partial amino acid sequence of this subunit, its gene will be cloned. A mutational analysis of this gene will determine the importance of proofreading during DNA replication of this eucaryote. Other mechanisms that lead to a high overall fidelity of DNA duplication will also be investigated. The yeast replication fork will be studied in vivo using pulse labeling methods to determine whether DNA replication in yeast is discontinuous or semidiscontinuous. The proposed study of eukaryotic replication will focus on yeast because, of all eukaryotic organisms, yeast is the one most amenable to biochemical and genetic manipulation. An intensive study of DNA replication and its regulation in yeast will undoubtedly be of significance in understanding cell growth and division in higher organisms. In addition, a better understanding of the mechanisms that induce or prevent mutations in yeast may lead to a better understanding of similar mechanisms in humans.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physiological Chemistry Study Section (PC)
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Washington University
Schools of Medicine
Saint Louis
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Burgers, Peter M J; Kunkel, Thomas A (2017) Eukaryotic DNA Replication Fork. Annu Rev Biochem 86:417-438
Koc, Katrina N; Singh, Saurabh P; Stodola, Joseph L et al. (2016) Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase ?. Nucleic Acids Res 44:3811-9
Burgers, Peter M J; Gordenin, Dmitry; Kunkel, Thomas A (2016) Who Is Leading the Replication Fork, Pol ? or Pol ?? Mol Cell 61:492-493
Stodola, Joseph L; Stith, Carrie M; Burgers, Peter M (2016) Proficient Replication of the Yeast Genome by a Viral DNA Polymerase. J Biol Chem 291:11698-705
Stojkovi?, Gorazd; Makarova, Alena V; Wanrooij, Paulina H et al. (2016) Oxidative DNA damage stalls the human mitochondrial replisome. Sci Rep 6:28942
Stodola, Joseph L; Burgers, Peter M (2016) Resolving individual steps of Okazaki-fragment maturation at a millisecond timescale. Nat Struct Mol Biol 23:402-8
Kochenova, Olga V; Bezalel-Buch, Rachel; Tran, Phong et al. (2016) Yeast DNA polymerase ? maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations. Nucleic Acids Res :
Cho, Jang-Eun; Huang, Shar-Yin N; Burgers, Peter M et al. (2016) Parallel analysis of ribonucleotide-dependent deletions produced by yeast Top1 in vitro and in vivo. Nucleic Acids Res 44:7714-21
Wanrooij, Paulina H; Burgers, Peter M (2015) Yet another job for Dna2: Checkpoint activation. DNA Repair (Amst) 32:17-23
Sparks, Justin L; Burgers, Peter M (2015) Error-free and mutagenic processing of topoisomerase 1-provoked damage at genomic ribonucleotides. EMBO J 34:1259-69

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