Development of a Specialized Yeast Strain to Monitor Recruitment Patterns of Saw1 Mediated Rad1‐Rad10 Recruitment to DNA Damage Sites through Single‐Strand Annealing

Damage to the structural integrity of DNA by exposure to ionizing radiation is the cause of many mutations that accumulate in the human genome, which can lead to cancer and aging. The DNA double‐strand break repair (DSBR) pathways limit this accumulation by continuously repairing such damage as it forms. Single‐strand annealing (SSA) is one mode of DSB repair that occurs in regions that have tandem arrays of DNA repeats. Upon DSB induction, the processing of broken ends exposes complementary single‐stranded regions that anneal and produce 3′ overhanging flaps deriving from the sequence originally situated between the repeats. This DNA flap structure recruits Rad1‐Rad10 endonuclease to cleave the overhanging 3′ DNA. Although recent literature reported that the Saw1 protein mediates recruitment of Rad1‐Rad10 when flaps are 500 deoxynucleotides long but not ~10, the minimum flap length for Saw1 to recruit Rad1‐Rad10 in vivo is unknown. The ultimate aim of this project is to determine whether Saw1 is required to recruit Rad1‐Rad10 to SSA sites when 3′ overhanging flaps are slightly longer (~20 deoxynucleotides) . To address this important question, we have prepared a specific yeast strain containing an inducible DSB site flanked by DNA repeats that will yield 20 bp overhang flaps upon DSB induction and repair by SSA. The strain will additionally contain a fluorescently labeled Rad10 gene (Rad10‐YFP) and DSB site (DSB‐RFP) to monitor Rad10 localization to DSB sites by fluorescence microscopy in the presence or absence of Saw1. Cloning of the 20 bp flap yeast strain was achieved by constructing two DNA plasmids that each contained the regions required to derive the desired substrate. The two plasmids were linearized and transformed into a progenitor yeast strain. Resulting transformants were screened by PCR to confirm integration of the recombinant DNA cassettes; an I Sce ‐I restriction site situated between two homologous HIS3 DNA repeats however spaced away from each other by 10 nonhomologous bp. DNA sequencing and I Sce ‐I restriction digest further supported proper insertion of the DNA cassettes and functionality of the inducible DSB site necessary for microscopy. This specialized yeast strain will be able to be used as an SSA substrate in consequent fluorescent microscopy experiments and will determine the necessity of Saw1 in recruiting Rad1‐Rad10 to SSA sites bearing ~20 deoxynucleotide flaps. Support or Funding Information The authors thank NIH grant (SC3GM093858) for funding.