This is a proposal to examine kinetic parameters that influence the DNA replication fidelity of DNA polymerase. Three polymerases will be examined: T4 DNA polymerase, E. coli Pol II, and E. coli Pol III. Two new assays developed by the P.I. will be used to determine the influence of DNA sequence context and DNA polymerase accessory proteins on misincorporation frequencies opposite normal bases and opposite abasic sites. In addition, the mechanism of switching between polymerization and excision will be examined. Dr. Goodman studies the detailed mechanism of action of DNA polymerases, principally relating to mispairing in spontaneous mutagenesis and replication of AP sites. He lists three Specific aims: (1) Three fidelity reactions (nucleotide insertion, proofreading and extension) will be measured for Pol III (in various forms: a subunit, core and holoenzyme) to understand the molecular basis of mispairing. (2) Wild type and mutant versions of T4 Pol and Pol II will be used to study switching between polymerase and proof reading active sites. (3) The ability of accessory proteins that enhance processivity to affect bypass of AP sites will be assessed. (4) A pre-steady state kinetic assay (fluorescence of 2-aminopurine) will be used to study in real-time various aspects of polymerase reaction mechanism.
| Pham, Phuong; Afif, Samir A; Shimoda, Mayuko et al. (2017) Activation-induced deoxycytidine deaminase: Structural basis for favoring WRC hot motif specificities unique among APOBEC family members. DNA Repair (Amst) 54:8-12 |
| Petruska, John; Goodman, Myron F (2017) Relating DNA base-pairing in aqueous media to DNA polymerase fidelity. Nat Rev Chem 1: |
| Pham, Phuong; Afif, Samir A; Shimoda, Mayuko et al. (2016) Structural analysis of the activation-induced deoxycytidine deaminase required in immunoglobulin diversification. DNA Repair (Amst) 43:48-56 |
| 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 |
| Senavirathne, Gayan; Bertram, Jeffrey G; Jaszczur, Malgorzata et al. (2015) Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution. Nat Commun 6:10209 |
| Mak, Chi H; Pham, Phuong; Afif, Samir A et al. (2015) Random-walk enzymes. Phys Rev E Stat Nonlin Soft Matter Phys 92:032717 |
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