In this project we investigate the mechanisms of DNA replication fidelity in E. coli by a combination of in vivo and in vitro approaches. In vivo, we investigate the specificity of mutation in the E. coli Lat gene in strains affected in various aspects of replication fidelity. For example, analysis of sequenced lacI mutations in wild-type, mismatch repair defective mutL strains, and proofreading defective mutDmutL strains, has allowed estimates to be made for the efficiencies and specificities of in vivo base selection, exonucleolytic proofreading and DNA mismatch repair. In vitro, we have developed novel in vitro fidelity assays, again using the lacI gene as a target, allowing measurement of the fidelity properties of purified DNA polymerase III in its various assemblies, ranging from the isolated alpha subunit to the complete holoenzyme. Results so far indicate that the fidelity behavior of polymerase III in vitro is quite distinct from that in vivo. For example, DNA polymerase III in vitro has a high propensity for producing frameshift mutations and a low propensity for making base-pair substitutions, which is exactly the opposite of what occurs in vivo. The possible reasons behind these differences are currently being investigated.
| Maslowska, Katarzyna H; Makiela-Dzbenska, Karolina; Mo, Jin-Yao et al. (2018) High-accuracy lagging-strand DNA replication mediated by DNA polymerase dissociation. Proc Natl Acad Sci U S A 115:4212-4217 |
| Babu, Vignesh M P; Itsko, Mark; Baxter, Jamie C et al. (2017) Insufficient levels of the nrdAB-encoded ribonucleotide reductase underlie the severe growth defect of the ?hda E. coli strain. Mol Microbiol 104:377-399 |
| Tse, Lawrence; Kang, Tina Manzhu; Yuan, Jessica et al. (2016) Extreme dNTP pool changes and hypermutability in dcd ndk strains. Mutat Res 784-785:16-24 |
| Maslowska, Katarzyna H; Makiela-Dzbenska, Karolina; Fijalkowska, Iwona J et al. (2015) Suppression of the E. coli SOS response by dNTP pool changes. Nucleic Acids Res 43:4109-20 |
| Swerdlow, Sarah J; Schaaper, Roel M (2014) Mutagenesis in the lacI gene target of E. coli: improved analysis for lacI(d) and lacO mutants. Mutat Res 770:79-84 |
| Gawel, Damian; Fijalkowska, Iwona J; Jonczyk, Piotr et al. (2014) Effect of dNTP pool alterations on fidelity of leading and lagging strand DNA replication in E. coli. Mutat Res 759:22-8 |
| Ahluwalia, Deepti; Schaaper, Roel M (2013) Hypermutability and error catastrophe due to defects in ribonucleotide reductase. Proc Natl Acad Sci U S A 110:18596-601 |
| Schaaper, Roel M; Mathews, Christopher K (2013) Mutational consequences of dNTP pool imbalances in E. coli. DNA Repair (Amst) 12:73-9 |
| Ahluwalia, Deepti; Bienstock, Rachelle J; Schaaper, Roel M (2012) Novel mutator mutants of E. coli nrdAB ribonucleotide reductase: insight into allosteric regulation and control of mutation rates. DNA Repair (Amst) 11:480-7 |
| Fijalkowska, Iwona J; Schaaper, Roel M; Jonczyk, Piotr (2012) DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair. FEMS Microbiol Rev 36:1105-21 |
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