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.
NIH ExPorter
NIH ExPorter