The broad objective of this grant is to investigate how 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 coil, response to DNA damage is orchestrated by an operon, the """"""""SOS regulon"""""""", containing at least 43 different proteins under negative control of a repressor protein, LexA, and a multifunctional protein, RecA. In E. coil, and in animal cells as well, damage-induced DNA repair is aberrant. There is a reduction in fidelity, which 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. coll. Such repair depends on RecA protein interacting with a mutagenic UmuD'2C protein complex, which we showed, during the previous grant period, to be a new DNA polymerase, E. coil pol V. The discovery of this new polymerase provided the impetus for the """"""""explosive"""""""" growth in newly discovered eukaryotic polymerases having essential roles in the avoidance of skin cancer and in the generation of antibody diversity. There are numerous types of damage occurring in DNA when cells are exposed to chemicals, drugs or radiation. To study error-prone repair in vitro and in vivo, we have chosen to focus primarily on copying a site-directed abasic (apurinic/apyrimidinic) DNA lesion, a biologically relevant noncoding lesion that can occur by spontaneous and induced mechanisms. The absence of a coding base in DNA presents a strong block to replication. When replication past an abasic lesion does occur, it often results in a mutation. In this proposal, we will focus on the key biochemical interactions responsible for error-prone translesion DNA synthesis, involving pol V, RecA, single strand binding protein and polymerase processivity clamp proteins. We intend to determine the mechanisms governing targeting of repair polymerases to specific DNA lesions and mechanisms of trafficking between polymerases, to exchange a high fidelity replication polymerase blocked at a site of DNA damage with a low fidelity repair polymerase that can relieve the blockage at the expense of generating mutations. The data generated in the proposed experiments will have a major impact in explaining how damaged DNA is copied in prokaryotic and eucaryotic organisms.
| 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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