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 fidelity assays, again using the lacI gene as a target, allowing measurement of the fidelity of purified DNA polymerase III in its various (sub)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 in vitro produces an abnormally high level of (-1) frameshift mutations. This points to the existence of a previously undescribed in vivo fidelity system capable of preventing (-1) frameshifts and other mutations. This system is currently investigated by searching for E. coli mutants defective in this process. We have isolated novel mutants of the dnaX gene (encoding the tau subunit of HE) which specifically enhance frameshifts and transversion mutations, consistent with such a mechanism. We have also developed a system to measure, for the first time, the differences between leading and lagging strand replication on the E. coli chromosome. This system is being exploited to investigate the replication factors that are responsible for this strand-specific fidelity difference.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Intramural Research (Z01)
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U.S. National Inst of Environ Hlth Scis
United States
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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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