Arrest of replication fork progression evokes a concerted response in both prokaryotes and eukaryotes that is designed to repair whatever template damage that exists and restart replication. In bacteria, failure to restart replication is a lethal event. In eukaryotes, mutation of many of the proteins that are involved in replication fork repair leads to DNA damage syndromes and cancer predisposition. Thus, understanding the processes that occur at stalled replication forks, which is the goal of our studies, is of considerable significance. In the previous grant period, we made significant progress in elucidating the biochemical pathways of origin- independent loading of replisomes and recombination-dependent DNA replication, and discovered that the replisome has the inherent ability to prime the leading strand de novo, suggesting that replisomes may be able to restart replication downstream of leading-strand template damage. Many different events happen at stalled replication forks to prevent their becoming a source of genomic instability. Template damage can be repaired before or after resumption of replication, the replisome may be preserved, disassembled, or remodeled, the stalled replication fork itself can be remodeled, and replication has to be restarted. Intensive research from a number of laboratories during the past decade has addressed these issues, yet a unified description of what happens has yet to emerge. In the next grant period we propose to clarify these issues by reconstituting biochemically new reactions that will reveal how complicated processes such as replisome remodeling, replication fork remodeling, lesion repair and bypass, replication reactivation, and daughter-strand gap repair cooperate to achieve maintenance of genomic stability at stalled replication forks. We use in these investigations purified DNA replication, recombination, and repair proteins from Escherichia coli and specialized DNA templates containing DNA damage at specific sites.

Public Health Relevance

Every time a cell divides a complete and accurate copy of the genetic information stored in the chromosomes must be passed to each daughter cell. Failure to do so results in mutations that can cause disease. In this proposal we study the mechanisms that ensure that the chromosomes are copied completely by studying the enzymatic machinery that restarts the process of copying (DNA replication) if it stalls. The genes encoding a number of proteins involved in this repair process cause DNA damage syndromes when mutated and these mutated genes cause increased incidence of cancer in humans.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Reddy, Michael K
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Sloan-Kettering Institute for Cancer Research
New York
United States
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Nevin, Philip; Gabbai, Carolina C; Marians, Kenneth J (2017) Replisome-mediated translesion synthesis by a cellular replicase. J Biol Chem 292:13833-13842
Graham, James E; Marians, Kenneth J; Kowalczykowski, Stephen C (2017) Independent and Stochastic Action of DNA Polymerases in the Replisome. Cell 169:1201-1213.e17
Gupta, Sankalp; Yeeles, Joseph T P; Marians, Kenneth J (2014) Regression of replication forks stalled by leading-strand template damage: II. Regression by RecA is inhibited by SSB. J Biol Chem 289:28388-98
Gabbai, Carolina B; Yeeles, Joseph T P; Marians, Kenneth J (2014) Replisome-mediated translesion synthesis and leading strand template lesion skipping are competing bypass mechanisms. J Biol Chem 289:32811-23
Gupta, Sankalp; Yeeles, Joseph T P; Marians, Kenneth J (2014) Regression of replication forks stalled by leading-strand template damage: I. Both RecG and RuvAB catalyze regression, but RuvC cleaves the holliday junctions formed by RecG preferentially. J Biol Chem 289:28376-87
Gupta, Milind K; Guy, Colin P; Yeeles, Joseph T P et al. (2013) Protein-DNA complexes are the primary sources of replication fork pausing in Escherichia coli. Proc Natl Acad Sci U S A 110:7252-7
Yeeles, Joseph T P; Marians, Kenneth J (2013) Dynamics of leading-strand lesion skipping by the replisome. Mol Cell 52:855-65
Yeeles, Joseph T P; Poli, Jérôme; Marians, Kenneth J et al. (2013) Rescuing stalled or damaged replication forks. Cold Spring Harb Perspect Biol 5:a012815
Marceau, Aimee H; Bahng, Soon; Massoni, Shawn C et al. (2011) Structure of the SSB-DNA polymerase III interface and its role in DNA replication. EMBO J 30:4236-47
Yeeles, Joseph T P; Marians, Kenneth J (2011) The Escherichia coli replisome is inherently DNA damage tolerant. Science 334:235-8

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