Structure-Function Studies Of DNA Replication Fidelity

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Summary of Work: The long-term goals of this project are (1) to understand the fidelity and efficiency of the DNA replication and repair processes that control cell survival and genome stability in organisms from viruses to humans, and (2) to understand how environmental stress perturbs these processes, resulting in cytotoxicity, mutagenesis and adverse effects on human health. This year, progress was made towards understanding the functions of several different DNA polymerases, including replicative T7 DNA polymerase (family A), nuclear replicative pols alpha and delta (family B), DNA repair pols lambda and mu (family X), and translesion synthesis pol zeta (family B) and pols eta, kappa and iota (family Y). These studies are revealing the strategies used by cells to accurately replicate normal DNA as well as DNA damaged by environmental agents, in a manner that avoids mutagenesis and cytotoxicity and the accompanying adverse health consequences. This project has been combined with Z01-ES-65046. Several repair processes operate prior to replication to remove DNA damage generated by endogenous cellular metabolism or exposure to environmental stress. These repair processes provide undamaged substrates for efficient and highly accurate DNA replication. High replication fidelity depends on the ability of DNA polymerases to select correct rather than incorrect nucleotides for incorporation into DNA without adding or deleting nucleotides. This selectivity is a prime determinant of fidelity at the replication fork and also during DNA synthesis associated with repair. Exonucleolytic proofreading of mismatches can further increase DNA replication fidelity. When DNA damage is not repaired prior to replication, certain lesions can stall progression of the replication fork. Under these circumstances, cell cycle checkpoints, translesion synthesis reactions and recombinational repair processes operate to maintain genome stability and improve cell survival. This project investigates these replication fidelity processes by studying the relationships between the structures of A, B, X, Y and RT family DNA polymerases and their accessory proteins, their biochemical properties and their putative functions in vivo. A dual approach is used involving in vitro biochemical studies with in vivo genetic studies in yeast and mice and humans.