Exploring Rad1‐Rad10 Recruitment by Slx4 in Relation to Cell Cycle

Abstract

DNA damage, caused by various exogenous and endogenous agents such as ionizing radiation and replication stress, gives rise to mutations in DNA that can cause cancer. To combat this, eukaryotic cells use evolutionarily conserved repair mechanisms that mend the DNA. The choice of repair pathway depends primarily on the form of damage, its location within the genome, and the cell cycle phase. For example, single‐strand annealing (SSA), a form of DNA double‐strand break repair (DSBR), is favored in regions between DNA repeats while synthesis‐dependent strand annealing (SDSA), another DSBR mode, needs sister chromatids which act as the template DNA for repair. In order for SSA and SDSA to be completed, the Rad1‐Rad10 endonuclease cleaves overhanging flaps that result from the annealing step before the final ligation can proceed.We have preliminary data indicating that Slx4, a protein present in S. cerevisiae (baker’s yeast), is partially required in the recruitment of Rad1‐Rad10 to repair sites during cell division (S, G2 and M phases). However, cells characterized as being in cell division were not differentiated during data analysis due to the size of the population within those phases. Our research seeks to elucidate the phase in which Slx4 exerts its recruitment properties by repeating our earlier fluorescence microscopy recruitment assay using yeast strains that contain fluorescently‐tagged proteins for monitoring both the colocalization of Rad1‐Rad10 to an induced double‐strand break site and the phase of the cell cycle in cell cycle‐synchronized cultures.To help us clearly distinguish between cell cycle phases during fluorescence microscopy, we have integrated a fluorescently labeled spindle pole body protein (Spc110‐CFP) into our strains because of its dramatic morphology shifts during cell division. Yeast genetic crosses were conducted to combine Spc110‐CFP with SLX4 mutant genes as well as other features that will allow us to perform fluorescence microscopy and flow cytometry analysis in a single strain. Following cell cycle arrest by alpha‐factor, > 90% of cells showed a single Spc110‐CFP focus, indicating that most cells were successfully arrested at the G1/S phase boundary, but flow cytometry analysis showed that only about 60% of cells contained a DNA content consistent with G1/S arrest. These data indicate that cell cycle arrest and its monitoring by the appearance of Spc110‐CFP foci are effective but further efforts are needed to optimize the protocol. Along these lines, we began creating a new SLX4 deletion mutant using Polymerase Chain Reaction (PCR) to amplify a transplacement mutant gene in which the SLX4 coding region has been swapped with the antibiotic marker NATMX4. The final PCR product was A‐tailed and then ligated into the pGEMT‐Easy vector. E. coli transformants were screened by PCR for the insert and will be sequenced to verify the SLX4 region is in the plasmid before moving forward with yeast genetic crosses.Support or Funding InformationNational Institute of Health (NIH) (Grant SC1GM127204), NIH MARC Program (NIH Grant GM008395), California State University, Northridge College of Science and Math, California State University, Northridge Department of Chemistry and Biochemistry