The repair of DNA damage is crucial to survival of all organisms. Thus, it is rfot surprising that the major DMA damage repair pathways, such as nucleotide excision repair and mismatch repair, are conserved from bacteria to man. These pathways are efficient and, for the most part, do not require the chromosomal DNA replication machinery for their activity. How do cells deal with the encounter between a replication fork and template DNA damage? What happens when the damage itself inactivates the replication fork?, creating a requirement for both replication fork restart and repair of the damage. As a result of studies from a number of groups, many centered, as ours have been, on the properties of PriA and its gene, a new paradigm has emerged describing the replication of the bacterial chromosome. This paradigm holds that even under normal growth conditions, the replication forks formed at oriC become inactivated as a result of an encounter with endogenous DNA template damage. This creates a requirement for both repair of the damage and reactivation of the replication forks. Our studies in the previous grant period have demonstrated that the <)>X174-type primosome is required for replication fork reactivation where it directs the assembly of a new replication fork on DNA substrates that are generated by the action of the recombination proteins. Furthermore, genetic data suggests that there are multiple pathways of replication fork restart involving different combinations of the primosomal proteins. In order to understand completely this intersection of two of the major pathways of DNA metabolism, we will model replication fork demise and reactivation in vitro. We will proceed by asking the following questions: What is the fate of the enzymatic components of the replication fork after a collision with either template DNA damage or a frozen protein-DNA complex? How is replication fork demise affected by the location and type of damage to the DNA? What are the DNA structures left at stalled replication forks? What conditions lead to DNA breakage at stalled replication forks? What is the biochemical basis for the existence of multiple pathways of replication fork restart? And, does the manner of recombination protein-directed processing of the DNA at a stalled fork direct the enzymatic pathway of replication forkreactivation? Using purified recombination and replication proteins, we will study the demise and reactivation of replication forks formed in isolated replisome complexes at oriC on small plasmid minichromosomes that have been engineered to carry specific types of DNA damage in specified locations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM034557-27
Application #
7747960
Study Section
Special Emphasis Panel (NSS)
Program Officer
Hagan, Ann A
Project Start
1984-07-01
Project End
2010-12-31
Budget Start
2010-01-01
Budget End
2010-12-31
Support Year
27
Fiscal Year
2010
Total Cost
$670,898
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
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
Gabbai, Carolina B; Marians, Kenneth J (2010) Recruitment to stalled replication forks of the PriA DNA helicase and replisome-loading activities is essential for survival. DNA Repair (Amst) 9:202-9

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