The overall goal of NIGMS-funded research in my lab is to describe the molecular and cellular mechanisms by which cells sense the presence of DNA damage and carry our repair of chromosomal double-strand breaks (DSBs). Using budding yeast as a model system, it is possible to induce site-specific DSBs with a high degree of synchrony not yet possible in mammalian cells, allowing ?in vivo biochemistry? approaches to monitor intermediate steps in DSB repair and DNA damage signaling. The first of three foci of this proposal investigates key questions concerning DSB repair by homologous recombination. How are homologous donor sequences found and used to repair a DSB? How are mismatches tolerated and repaired during different steps of recombination? How do cells deal with chromatin during repair and how is chromatin re- established after repair is complete? And how is gene editing accomplished using single-stranded DNA templates? The second area seeks to understand what is the basis of the 1000-fold increase in mutations associated with DSB repair and how microhomologies are used in repair-dependent template switching, creating complex chromosome rearrangements analogous to events recently found in human cancers. The third main objective is to understand how the DNA damage checkpoint and DNA damage-induced autophagy are regulated. We wish to determine: How does the DNA damage response affect DSB repair? How is the DNA damage checkpoint maintained and how is autophosphorylation of Mec1ATR regulated? What is the basis of a regulatory hand-off between the damage response and the spindle assembly checkpoint? Finally, we will determine the targets of DNA damage-induced autophagy and how autophagy contributes to cell cycle arrest in response to even a single unrepaired DSB. These studies will provide new insights and guidance in defining the DSB repair and checkpoint signaling in human cells.
Cancer cells that display profound genome instability have lost their ability to repair chromosome double-strand breaks (DSBs) by accurate processes of homologous recombination and also have lost their ability to activate the DNA damage response, which arrests cell cycle progression. This proposal investigates the mechanisms of repairing DSBs by homologous recombination and the interrelationship between DNA repair and the DNA damage response, using budding yeast, where it is possible to induce site-specific DNA damage with high synchrony not yet possible in mammalian cells. Based on the strong evolutionary conservation of core components of DNA repair and DNA damage checkpoint, insights from this model organism will provide important guidance to investigating these processes in human cells.
