of Work: 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 lacI 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 (HE). Interestingly, the fidelity behavior of polymerase III in vitro is quite different from that in in vivo. Specifically, DNA polymerase III (a subunit or HE) in vitro produces an abnormally high level of (-1) frameshift mutations. These data point to the existence of a novel fidelity system in vivo that prevents (-1) frameshift mutations. This system is currently investigated by isolating E. coli mutants defective in this process. As part of this effort we have isolated novel mutants of the dnaX gene (encoding the t subunit of HE) that specifically enhance frameshifts and transversion mutations. We have also demonstrated a mutator effect for an existing dnaN mutant, encoding the pol III processivity factor (sliding clamp). Finally, we have developed a system to measure for the first time the differences between leading and lagging strand replication on the E. coli chromosome.
| 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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