Molecular mechanisms of DNA polymerase fidelity

Faithful replication of the genome is carried out at the molecular level by DNA polymerases. The ability of these polymerases to efficiently and accurately copy the genome is critical to life as we know it. The goal of this thesis has been to examine and characterize mechanisms that contribute to the ability of DNA polymerases to properly replicate DNA. Traditional fidelity models, based on hydrogen bonding of Watson-Crick nucleobases, do not completely account for the high replication fidelity of DNA polymerases. I hypothesized that additional biophysical factors that contribute to the fidelity of DNA synthesis could be revealed and/or characterized by examining the polymerase activity kinetically when it is presented with modified or non-natural DNA template bases or nucleotides. Furthermore, because polymerase activity is regulated in part by its interactions with other replication fork proteins, I further hypothesized that these replication fork components could also influence fidelity.; The data in Chapter 3 indicate that bacteriophage T4 DNA polymerase interaction with its “clamp” processivity factor does not facilitate the ability of the polymerase to carry out translesion synthesis (TLS) when it encounters an abasic lesion in the DNA template strand. Kinetic evaluation of the residence time of the polymerase and clamp (replicase) at the lesion suggests that the polymerase is carrying out an idle turnover process while “stalled” at the abasic lesion.; Chapter 4 examines non-hydrogen bonding physicochemical parameters of nucleobases that are predominantly involved in replication fidelity and efficiency. The data show that adding size to normal nucleobases modestly improves the rates of their incorporation opposite an abasic template lesion. However, improvement of the base-stacking ability of a nudeobase is shown to improve its incorporation efficiency markedly. I have identified a nucleobase that incorporates with high efficiency opposite damaged DNA, and acts as a chain terminator, preventing further DNA synthesis.; In Chapter 5, I used principles of rational drug design to identify and characterize two novel nucleobases that DNA polymerase can utilize to form an artificial base pair. This nucleobase pair has the potential to expand the genetic code, leading to applications in biotechnology and research.