Mechanisms Of E. coli Replication Fidelity

National Institute Of Environmental Health Sciences

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Abstract

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 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 polymerase dissociation occurring in this strand as an additional fidelity system. We have also shown 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, enzymes lacking an exonucleolytic proofreading activity. Access by Pol II is largely error-free, and serves to exclude access by Polymerases IV and V. Studies of the role of the dNTP levels in E. coli cells, using strains with altered dNTP levels, like the ndk and dcd deficient strains, have revealed positive correlations between the bacterial replication error rates and the particular dNTP pool disturbances, both in terms of absolute dNTP levels and the incorrect/correct dNTP ratios. Low overall dNTP levels were found to be critical to the efficient functioning of the exonucleolytic proofreading mechanism. A novel set of E. coli mutator strains was isolated by altering the allosteric regulation of the enzyme ribonucleotide reductase, which plays a critical role in the synthesis of the cellular dNTPs. We have also discovered an inhibitory effect of dNTP pool changes in ndk and dcd mutants on the ability of E. coli to express the error-prone SOS response, likely through an adverse effect on the stability and/or activity of the RecA nucleofilament.