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.
| Petruska, John; Goodman, Myron F (2017) Relating DNA base-pairing in aqueous media to DNA polymerase fidelity. Nat Rev Chem 1: |
| Goodman, Myron F (2016) Better living with hyper-mutation. Environ Mol Mutagen 57:421-34 |
| Jaszczur, Malgorzata; Bertram, Jeffrey G; Robinson, Andrew et al. (2016) Mutations for Worse or Better: Low-Fidelity DNA Synthesis by SOS DNA Polymerase V Is a Tightly Regulated Double-Edged Sword. Biochemistry 55:2309-18 |
| Oertell, Keriann; Harcourt, Emily M; Mohsen, Michael G et al. (2016) Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity. Proc Natl Acad Sci U S A 113:E2277-85 |
| Goodman, Myron F; McDonald, John P; Jaszczur, Malgorzata M et al. (2016) Insights into the complex levels of regulation imposed on Escherichia coli DNA polymerase V. DNA Repair (Amst) 44:42-50 |
| Robinson, Andrew; McDonald, John P; Caldas, Victor E A et al. (2015) Regulation of Mutagenic DNA Polymerase V Activation in Space and Time. PLoS Genet 11:e1005482 |
| Gruber, Angela J; Erdem, Aysen L; Sabat, Grzegorz et al. (2015) A RecA protein surface required for activation of DNA polymerase V. PLoS Genet 11:e1005066 |
| Goodman, Myron F (2014) The discovery of error-prone DNA polymerase V and its unique regulation by RecA and ATP. J Biol Chem 289:26772-82 |
| Erdem, Aysen L; Jaszczur, Malgorzata; Bertram, Jeffrey G et al. (2014) DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent ATPase. Elife 3:e02384 |
| 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 |
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