DNA damage can arise from a multitude of exogenous factors (such as ultraviolet radiation) and endogenous factors (such as replication fork collapse). One type of DNA damage is the double stranded break (DSB), which when left unrepaired can lead to cell death and cancer. There are two major DSB repair pathways that operate within yeast and mammalian cells: non-homologous end joining and homologous recombination (HR). Resection, the conversion of dsDNA to ssDNA through nuclease activities, is an essential step in the initiation of the HR pathway and is required to carry out the search for a homologous (template) strand for repair. Further, resection of the dsDNA around the DSB commits the lesion to repair by HR. Our objective was to develop an assay in the model organism S. pombe to simultaneously measure the extent of resection and DSB position within the nucleus. Towards this goal, we developed and hope to validate a rapid DSB induction system based on a chimeric human estrogen receptor hormone binding domain (ER) and the site-specific endonuclease Sce-1. An Sce-1 cut site was engineered next to a lacO array, which was visualized by expression of lacI GFP. We predict that the estrogen receptor-SceI chimera can access the nucleus only upon addition of the small molecule, estradiol, which releases the ER from the endogenous factor Hsp90 that sequesters the chimera in the cytoplasm. Using this system, we will be able to visualize the DSB in live cells after addition of estradiol, translocation of ER-SceI into the nucleus and induction of the DSB. Further, when resection takes place, the dsDNA is converted to ssDNA, which then evicts the dsDNA binding lacI-GFP. Our immediate goal is to measure the extent of resection and determine the position of the DSB. Further, we hypothesize that a DSB break will move from the interior to the periphery of the nucleus when it is extensively resected. We also predict that the state of chromatin (heterochromatin found in the periphery vs. euchromatin found in the interior) will have an effect on the amount of resection that can occur when the lacO array/SceI site is integrated within these different chromatin landscapes. Our long-term goal is to determine how these two parameters, extent of resection and nuclear position, together help to determine the pathway used to repair a DSB.

Rapid fire synchronous induction of double strand break infission yeast

247th National Meeting (2014)

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