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 proposed a model in which tau subunit may act as a sensor for replication errors, particularly those that may lead to a (temporarily) stalled replication complex. In the absence of this sensor function (in certain dnaX mutants), HE may be forced to extend certain mismatches, leading to enhanced production of transversions and defined (-1) frameshifts. We have also developed a system to measure the differences between leading and lagging strand replication on the E. coli chromosome. Our results with this system suggest that the lagging strand is synthesized more accurately than the leading strand, presumably because of the availability of an additional fidelity system not available in the leading strand. We have also demonstrated that the lagging strand is more readily accessible for the E. coli accessory DNA polymerases, such as Pol II, Pol IV, and Pol V. This access may lead to a mutator effect in the case of polymerases IV and V, which are enzymes lacking an exonucleolytic proofreading activity. However, access by Pol II is largely error-free, and serves to exclude access by Polymerases IV and V. Detailed analysis of the dnaX36 mutator mutant has suggested that part of the mutator activity is due to Pol IV but that Pol II plays a very large role in proofreading errors made by Pol III in this mutant. Pol I also plays a role in determining chromosomal replication fidelity. However, its role is limited to the faithful filling of the Okazaki fragment gaps in the lagging strand.

Project Start
Project End
Budget Start
Budget End
Support Year
18
Fiscal Year
2013
Total Cost
$327,745
Indirect Cost
City
State
Country
Zip Code
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
Taira, Kentaro; Kaneto, Satomi; Nakano, Kota et al. (2013) Distinct pathways for repairing mutagenic lesions induced by methylating and ethylating agents. Mutagenesis 28:341-50
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
Conte, Emanuele; Vincelli, Gabriele; Schaaper, Roel M et al. (2012) Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core. Arch Biochem Biophys 523:135-43
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

Showing the most recent 10 out of 13 publications