Investigating SLX4‐Dependent Recruitment of a DNA Repair Protein to DNA Double Strand Breaks in Dividing Cells

Abstract

Cancer is a disease that functions as a consequence of abnormal cell growth, generally caused by DNA damage that triggers abnormal cell function. One form of DNA damage is the double‐strand break (DSB) in which both strands of a chromosome are severed in near proximity. In normal cells, several forms of DSB repair, including single strand annealing (SSA) and synthesis dependent strand annealing (SDSA), fix the double strand break (DSB), restoring an intact chromosome, albeit sometimes with an altered DNA sequence. These DNA repair processes require the sequential action of numerous proteins that enable their many steps. In S. cerevisiae these include the Rad1‐Rad10 endonuclease complex, which cleaves nonhomologous DNA flaps in SSA and SDSA as one of the final steps.We have evidence that the Slx4 protein is partially required for the recruitment of Rad1‐Rad10 to DSB sites when cells are dividing (in S, G2 or M phases). Those experiments did not show the basis for the “partial” nature of the recruitment, but our hypothesis is that the Slx4‐dependency is tied to a specific phase of the cell cycle, possibly S phase. A plausible explanation is that when Slx4 exercises its checkpoint signal dampening role in S phase, which promotes homologous recombination, it indirectly recruits Rad1‐Rad10 to double‐strand break repair sites. Since, following DNA damage, checkpoint signal dampening is triggered (in part) by phosphorylation of Slx4 in S phase and the specific phosphorylation sites for this are known, we will test our hypothesis by creating mutant strains which lack these sites and repeating the earlier experiments using synchronized cell cultures. We began creating the mutant strains by cloning DNA plasmids containing the slx4‐7MUT gene, our focus, in which wild‐type SLX4 has been mutated to alanine at the seven phosphorylation sites activating checkpoint signal dampening. Candidate clones were screened by PCR and DNA sequencing. The successful candidate plasmids were transformed into a panel of W303 background yeast strains and integration of the mutant cassettes was confirmed by PCR and DNA sequencing screens. The strains were then crossed to strains bearing the fluorescence microscopy assay markers we will use in the fluorescence microscopy assay being employed. We have carried out preliminary fluorescence microscopy assays in which cultures of the progeny of these crosses were synchronized at the G1/S boundary and released back into the cell cycle that show acceptable arrest but the limited release back into the cell cycle that was observed requires further optimization. Using this separation‐of‐function mutant strain will allow us to compare recruitment of Rad1‐Rad10 in SLX4 wild‐type strains with Slx4 checkpoint signal dampening mutants. Understanding the process of Rad1‐Rad10 recruitment may aid in the design and development of a more effective means of cancer treatment.