The broad objectives of this grant are to investigate biochemical mechanisms by which damaged DNA is copied by the cell's replication and repair enzymes, focusing on proteins that are induced in response to DNA damage. Damage-induced DNA repair occurs in both procaryotic and eucaryotic organisms. In Escherichia coli, response to DNA damage is orchestrated by an operon, the "SOS regulon", containing more than 40 different proteins under negative control of a repressor protein, LexA, and a multifunctional protein, RecA. In E. coli, and in animal cells, damage-induced DNA repair can often be aberrant. There is a reduction in fidelity that enables replication to continue past blocking DNA damage sites. The primary goal of this proposal is to elucidate the biochemical basis for SOS-induced error-prone repair in E. coli. Such repair depends on RecA protein interacting with a mutagenic UmuD'2C protein complex, which we showed to be a new DNA polymerase, E. coli pol V. The discovery of this new polymerase provided the impetus for the "explosive" growth based on subsequent discoveries of fundamentally important error- prone eukaryotic DNA repair polymerases involved, for example, in avoiding skin cancer and in generating antibody diversity. There are numerous types of damage occurring in DNA when cells are exposed to chemicals, drugs or radiation. To study the biochemical basis of error-prone repair in vitro, we have chosen to focus primarily on copying site-directed biologically relevant lesions that occur spontaneously or from exogenous DNA damage. DNA template lesions often present a strong block to DNA replication. When replication past a lesion does occur, it can generally cause a mutation targeted to the DNA damage site. In this proposal, we will focus on the key biochemical interactions responsible for error-prone translesion DNA synthesis, involving E. coli DNA polymerase V, RecA protein, and polymerase processivity clamp proteins. We intend to determine the mechanisms governing targeting of repair polymerases to damaged DNA and mechanisms of trafficking between polymerases, to exchange high fidelity replication polymerase blocked at a site of DNA damage with low fidelity repair polymerases that can relieve the blockage at the expense of generating mutations. During the previous grant period, we discovered an unprecedented mechanism in DNA replication by which E. coli DNA polymerase V is unable to copy damaged or undamaged DNA unless activated in trans by RecA bound to ssDNA not being copied by pol V. A central theme of this proposal is to establish the mechanistic basis for RecA- ssDNA transactivation of pol V. The data generated in the proposed experiments will have major biological impact in exploring the principles of how damaged DNA is copied. Project Narrative: In organisms ranging from bacteria to humans, almost all mutations are deleterious, serving as a root cause of numerous diseases including cancer. Ironically, however, it has recently been found that there are error-prone DNA repair pathways that are beneficial, indeed essential, in providing immunological diversity, general fitness and avoidance of cell death, and even protecting against some human diseases, for example skin cancer. The proposed research explores the biochemical basis governing the ability of error-prone DNA polymerases to copy damaged DNA that would otherwise cause a cessation of chromosome replication leading to cell death.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES012259-23
Application #
8230493
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Shaughnessy, Daniel
Project Start
1989-07-01
Project End
2013-08-31
Budget Start
2012-03-01
Budget End
2013-08-31
Support Year
23
Fiscal Year
2012
Total Cost
$359,452
Indirect Cost
$138,929
Name
University of Southern California
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Vaisman, Alexandra; McDonald, John P; Noll, Stephan et al. (2014) Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli. Mutat Res 761:21-33
Donigan, Katherine A; McLenigan, Mary P; Yang, Wei et al. (2014) The steric gate of DNA polymerase ? regulates ribonucleotide incorporation and deoxyribonucleotide fidelity. J Biol Chem 289:9136-45
Corzett, Christopher H; Goodman, Myron F; Finkel, Steven E (2013) Competitive fitness during feast and famine: how SOS DNA polymerases influence physiology and evolution in Escherichia coli. Genetics 194:409-20
Pomerantz, Richard T; Goodman, Myron F; O'Donnell, Michael E (2013) DNA polymerases are error-prone at RecA-mediated recombination intermediates. Cell Cycle 12:2558-63
Pomerantz, Richard T; Kurth, Isabel; Goodman, Myron F et al. (2013) Preferential D-loop extension by a translesion DNA polymerase underlies error-prone recombination. Nat Struct Mol Biol 20:748-55
Goodman, Myron F; Woodgate, Roger (2013) Translesion DNA polymerases. Cold Spring Harb Perspect Biol 5:a010363
Yamanaka, Kinrin; Minko, Irina G; Finkel, Steven E et al. (2011) Role of high-fidelity Escherichia coli DNA polymerase I in replication bypass of a deoxyadenosine DNA-peptide cross-link. J Bacteriol 193:3815-21
Patel, Meghna; Jiang, Qingfei; Woodgate, Roger et al. (2010) A new model for SOS-induced mutagenesis: how RecA protein activates DNA polymerase V. Crit Rev Biochem Mol Biol 45:171-84
Jiang, Qingfei; Karata, Kiyonobu; Woodgate, Roger et al. (2009) The active form of DNA polymerase V is UmuD'(2)C-RecA-ATP. Nature 460:359-63
Minko, Irina G; Yamanaka, Kinrin; Kozekov, Ivan D et al. (2008) Replication bypass of the acrolein-mediated deoxyguanine DNA-peptide cross-links by DNA polymerases of the DinB family. Chem Res Toxicol 21:1983-90

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