Structure-Function Studies Of DNA Replication Fidelity

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

Abstract

Summary: This year, our group investigated several subjects regarding DNA replication and its fidelity. One publication involved studying the repair of double strand DNA breaks by microhomology mediated end-joining conducted by DNA polymerases theta and delta. This pairing of polymerase delta with a polymerase capable of end-bridging synthesis, polymerase theta, may help to explain why the normally high-fidelity polymerase delta participates in genome destabilizing processes such as mitotic DNA synthesis and microhomology-mediated DNA break-induced replication. A second publication involved repair of double strand DNA breaks by non-homologous end joining conducted by a mutator variant of DNA polymerase lambda. DNA polymerases lambda and mu are the two major enzymes that participate in DNA double-strand break repair by the nonhomologous end-joining pathway. Both polymerases direct synthesis from one DNA end using a template derived from a second DNA end. In this way, they promote the NHEJ ligation step and minimize the sequence loss normally associated with this repair pathway. A third study showed that the rates of single base mutations across the yeast nuclear genome are strongly increased in a strain harboring a mutator variant of DNA polymerase alpha combined with a mutation that inactivates the 3´-5´exonuclease activity of DNA polymerase delta. The observations in this study are consistent with the hypothesis that during initiation of nuclear DNA replication, single-base mismatches made by naturally exonuclease-deficient DNA polymerase alpha are extrinsically proofread by DNA polymerase delta, such that in the absence of this proofreading, the mutation rate is strongly elevated. We suggest that the mutational signature of defective extrinsic proofreading could appear in human tumors, thereby driving caner. The fourth and fifth studies investigated DNA ligase 1, the enzyme that finalizes DNA replication and repair by catalyzing the joining of DNA nicks. One study showed results on the role of a conserved amino acid in ribonucleotide discrimination by high-fidelity DNA ligase 1 at each step of the ligation reaction. The other study investigated the role of a second amino acid in assuring that DNA ligase 1 prevents formation of single base addition mutations at the end of replication. Both studies are potentially relevant to roles for DNA ligase 1 in preventing human diseases, including cancer and immunological diseases.