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Elucidation of Saw1 and Rad1–Rad10 Recruitment to Induced Single‐stand Annealing Sites as a Function of 3′ Flap Length
Author(s) -
Felix Juan Camberos,
Fregoso Fred,
Odango Rowen,
Iannolo Bailey,
Fischhaber Paula
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.800.1
Subject(s) - biology , dna repair , dna , homologous recombination , endonuclease , saccharomyces cerevisiae , microbiology and biotechnology , genetics , yeast
Unrepaired DNA damage leads to cancer and aging. A more detailed understanding of DNA damage repair mechanisms, specifically of double‐strand breaks (DSB's), can lead to new cancer prevention and treatment strategies. Two major DSB repair pathways include Synthesis‐Dependent Strand Annealing (SDSA), a conservative pathway resulting in perfect repair, and Single‐Strand Annealing (SSA), repair characterized by the loss of genetic material. During SSA in S. cerevisiae , complementary strands deriving from DNA repeats flanking a DSB become annealed, creating non‐homologous 3′ overhanging DNA flaps that are cleaved by the Rad1–Rad10 protein complex endonuclease activity. Prior research shows Saw1 is required to recruit Rad1–Rad10 to the DSB. However, recent studies show Rad1–Rad10 recruitment is Saw1‐independent if the overhanging flaps to be cleaved are short leaving ill‐defined the precise flap length triggering Saw1‐dependent Rad1–Rad10 recruitment. To elucidate the flap length at which Rad1–Rad10 requires Saw1 for recruitment during SSA, DSB's will be triggered by restriction digest in fluorescently labeled yeast strains containing a fluorescently‐labeled I‐ SceI cut site flanked by a variable number of non‐homologous base pairs (bp) between two DNA repeats. Repair sites containing variable length overhanging flaps produced by SSA will be monitored for recruitment of Rad1–Rad10 via fluorescence microscopy in pairs of strains that are either wild‐type in SAW1 or SAW1 deletion mutants. The specialized yeast strains needed are not available and the current goal of this project is their preparation. Strains are being prepared by integration of DNA cassettes bearing the I‐ SceI cut site flanked on either side by 20, 30 or 50 bp of non‐homologous DNA and the HIS3 gene. The 663 bp HIS3 gene provides the DNA repeat sequence. DNA plasmids bearing the cassettes were prepared by standard cloning methods. DNA for the 50 bp flap yeast strain has been successfully integrated into the yeast genome with PCR screens of yeast transformants showing incorporation of the correct DNA cassettes at the target chromosomal locus. Our 50 bp flap strain screen results show that the cloning scheme envisioned for the preparation of these specialized yeast strains is viable and preparation of one strain is complete. Support or Funding Information NIH‐SC3GM093858 and the Dr. Lan K. Wong Undergraduate Research Award.