Structure-Function Studies Of DNA Replication Fidelity

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This year, we published on several aspects of DNA synthesis fidelity. (1) We completed two studies on the fidelity of DNA synthesis by DNA polymerase lambda, an enzyme that participates in repairing DNA lesions resulting from exposure to environmental stress, including radiation. We identified and characterized a mutator variant polymerase missing a loop that interacts with the DNA template strand, and we obtained structures of this polymerase with either a matched or a G-T mismatched base pair at the active site. The mismatch has Watson-Crick geometry and is poised for catalysis, thus providing the first direct structural evidence to support Watson and Cricks 1953 proposal for one origin of spontaneous mutations. (2) We completed four studies on the incorporation of rNTPs into DNA during replication and its consequences. These consequences include replicative stress, and genome instability resulting from a novel mechanism involving strand cleavage by topoisomerase 1. (3) Using deep sequencing technology, we provided strong support for the idea that Pol delta is the primary lagging strand replicase for the whole nuclear genome in budding yeast. (4) We demonstrated that alterations in dNTP pools resulting from mutations in ribonucleotide reductase result in patterns of mutagenesis in vivo that are predicted by the first principles of replication fidelity that we and others established in vitro over the past two decades. Moreover, these patterns strongly suggest that certain dNTP pool imbalances reduce replication fidelity in vivo in a highly strand-specific manner. 5) Somewhat contrary to generally accepted logic, we demonstrated that Pol zeta can completely bypassing several different lesions in DNA templates without assistance from other polymerases. 6) We contributed to three collaborative studies by determining the fidelity of DNA synthesis by (a) Helicobacter pylori DNA polymerase 1, (b) by hybrid family A DNA polymerases engineered to more efficiently amplify ancient DNA samples, and (c) by Pols delta and epsilon as they copy dinucleotide repeat sequences that are highly abundant in mammalian genomes. We also collaborated to investigate inhibition of base excision repair catalyzed by DNA polymerase lambda. 7) We also published three reviews on DNA replication fidelity.