| DNA polymerases are the enzymes responsible for the vital task of faithfully duplicating genomes in order to pass on these genetically encoded instructions to their offspring. However, the process of faithfully propagating this information is hindered in all organisms due to endogenous and exogenous agents that damage DNA. While DNA repair mechanisms correct the vast majority of the resulting DNA lesions, unrepaired lesions do persist in the presence of fully functional repair mechanisms. Fortunately cells have evolved a class of promiscuous enzymes known as lesion bypass polymerases that have been shown to bypass DNA lesions that stall the high fidelity replicative DNA polymerases. Here, we have studied two DNA polymerases, human DNA polymerase lambda and Sulfolobus solfataricus DNA polymerase IV (Dpo4), which are thought to be involved in the previously mentioned cellular processes of DNA repair and DNA lesion bypass respectively.;In the process of establishing a minimal kinetic mechanism for the incorporation of a single nucleotide into undamaged DNA catalyzed by human DNA polymerase lambda, we discovered a novel mechanism in which one of its non-enzymatic N-terminal domains, the Proline-rich domain, dramatically increases the fidelity of the C-terminal DNA polymerase beta-like domain by 10- to 100-fold to the level equivalent to that observed with DNA polymerase beta, with which it shares 33% sequence identity. Moreover, we have also explored the effects of various structurally distinct DNA substrates on the catalytic efficiency of nucleotide incorporation where we determined the downstream strand and its 5'-phosphate increase the incorporation efficiency by 15- and 11-fold respectively.;We have used S. solfataricus Dpo4 as a model Y-family DNA polymerase to elucidate the kinetic mechanism for nucleotide incorporation at both 37°C and 56°C, demonstrating that Dpo4 uses an induced-fit mechanism to select and incorporate a correct nucleotide into undamaged DNA independent of reaction temperature. We have also demonstrated using a variety of techniques that Dpo4 predominantly uses two distinct pathways (A-rule and lesion loop-out mechanism) to bypass an abasic site lesion. Taken together, these observations provide compelling evidence for the observation made by Joyce and Benkovic that DNA polymerases defy a unified description. |
