Structure-Function Studies Of DNA Replication Fidelity

National Institute Of Environmental Health Sciences

Investigators

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Abstract

This year, our group investigated the following five subjects regarding DNA replication and its fidelity. (1) The first involves studying the effects deletion of the RRM3 and Pif1 helicases on replication fidelity. By genome-wide analysis, we showed that the mutational consequences due to loss of RRM3 vary depending on the genomic locus. Rrm3 promotes the accuracy of DNA replication across the yeast genome catalyzed by DNA polymerases delta and epsilon. This helicase is also important for preventing Pol zeta dependent mutagenesis at tRNA genes. We presented additional evidence that the sole human Pif1 family helicase in humans also promotes replication fidelity, suggesting that its role in mutation avoidance is evolutionarily conserved and may have potential tumor-suppressor function. (2) The second suject involves development of a method to monitor the stability of human mitochondrial DNA in a high throughput manner and using it to measure the rate of deletions in mitochondrial DNA isolated from wild type human tissues and in tissues harboring mutations in the mitochondrial DNA replicase, DNA polymerase gamma. (3) This year, we continued studies to measure the fidelity of the last step in completing replication of the lagging strand replication of the yeast nuclear genome, Okazaki fragment maturation. This process occurs by strand-displacement synthesis catalyzed by DNA polymerase delta, followed by removal of the resulting flapped DNA by Rad27 nuclease and the ending with ligation by DNA ligase 1 to complete lagging strand replication. We are currently investigating mutations generated by a DNA ligase point mutation in the active site that increases mutagenesis by at least four different enzymatic mechanisms. (4) We are investigating the role of extrinsic proofreading by DNA polymerase delta of replication errors made by DNA polymerase alpha during initiation of replicative DNA synthesis. (5) We are also investigating the role of the fourth B-family DNA, DNA polymerase zeta, in preventing mutations. Pol zeta is hypothesized to bypass lesions caused by environmental stress.