Biochemical and Genetic Studies of Genomic Stability

Citation

Fortini, Barbara Karmen Kraatz (2011) Biochemical and Genetic Studies of Genomic Stability. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ZH26-B677. https://resolver.caltech.edu/CaltechTHESIS:05172011-123048725

Abstract

Genomes face a constant barrage of threats from endogenous and exogenous sources. The need to maintain fidelity while replicating the entire genome during each cell division necessitates a dynamic cadre of proteins and protein complexes that participate in the DNA replication process. Furthermore, DNA can be damaged during all phases of the cell’s life and that damage must be recognized and repaired in a way that preserves genetic information. These studies focus on one enzyme at the nexus of DNA replication and DNA repair, the helicase/nuclease Dna2. We show that Dna2 possesses a novel ATP/Mn²⁺ dependent flap endo/exonuclease activity and a DNA end-independent endonuclease activity that is inhibited by Replication Protein A. The regulation of Dna2 activity in the context of the global DNA damage response is of great interest. To that end, we explored the relationship of Dna2 and the DNA damage sensor kinase Mec1. We find that Dna2 is phosphorylated by Mec1 following DNA damage in its N-terminal domain. We then extended these studies from yeast to higher eukaryotes utilizing the Xenopus cell free extract system. Using simulated double strand breaks (DSBs), we constructed a timeline of protein processing steps required for homologous recombination mediated repair. This strategy using Xenopus extracts also place Dna2 on chromatin during DNA replication, physically interacting with other proteins involved in lagging strand replication. Taken together, these biochemical and genetic studies elucidate the multiple roles the Dna2 enzyme plays in order to ensure genomic stability.

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