We are broadly interested in the mechanisms and energetics of DNA replication. Hallmarks of DNA replication include its high fidelity, typically resulting in one error for every 109 bases replicated, its regulation, and coordination between the continuous synthesis of the leading strand and generation of Okazaki fragments on the lagging strand.
A key unresolved question is how DNA polymerases (and RNA polymerases) discriminate between right and wrong dNTPs. The replicative DNA polymerases contribute significantly to this low error rate by only rarely incorporating wrong dNTPs, whereas the lesion bypass polymerases typically misincorporate dNTPs at much higher rates. We are addressing this question at two levels: how do DNA polymerases from different evolutionary families employ the chemical properties of a base to identify it as right and wrong, and what contributes to the large Δ(ΔG) between polymerization of right and wrong (d)NTPs?
We have recently developed an in vitro replication system employing purified herpes simplex virus 1 proteins and a minicircle template that recapitulates coupled leading and lagging strand synthesis. The proteins generate Okazaki fragments on the lagging strand and long leading strand products. Using this system in conjunction with studies on individual proteins (polymerase, helicase/primase, etc.) we are addressing the detailed mechanism of herpes DNA replication. This includes understanding the recycling of the herpes polymerase upon completion of an Okazaki fragment, the role of the herpes processivity factor in fidelity, how other essential herpes proteins impact replication, and the impact of different inhibitors on replication.