Construction of plasmids to make a specialized S. cerevisiae strain to investigate Saw1 protein recruitment in single‐strand annealing repair

Abstract

DNA damaging agents such as ionizing radiation and reactive oxygen species resulting from cellular metabolism can lead to double‐strand breaks (DSBs) which, if left unrepaired, may result in genetic diseases and cancer. Living systems are equipped with DNA repair pathways that alleviate such damage as it occurs. One mode of repair is single‐strand annealing (SSA), a non‐conservative pathway that utilizes DNA repeats flanking the DSB site. DSBs are resected 5′ to 3′ exposing complementary strands of the repeats which become annealed by the protein Rad52, generating overhanging 3′ flaps deriving from the DNA originally located between the repeats. Flap cleavage is achieved by the Rad1‐Rad10 endonuclease which is recruited by the protein Saw1. The point‐in‐time at which Saw1 recruits Rad1‐Rad10 is not known but likely occurs following annealing. To investigate this question, we are constructing a specialized yeast strain containing two inducible and fluorescently tagged DSB sites to monitor SSA by fluorescence microscopy. Simultaneous induction of both DSB sites will lead to repair by SSA that results in convergence of the two fluorescent signals during annealing by Rad52. Using this assay system, we will be able to peg Saw1 arrival to the annealing step, making it possible to detect whether Rad52 is required for the recruitment of Saw1 to DSB sites. An additional goal of this research project will be to investigate whether human Rad52 can be swapped in, in place of the endogenous yeast Rad52.The first step toward this goal has been to construct two DNA plasmids that will enable creation of a transgenic yeast strain with the features described above. The first plasmid will integrate one of the inducible DSB sites and one of the DNA repeats. Plasmid 1 was constructed by ligating into vector “pLAY500”: 1) a HYG marker from plasmid “pAAH1”, 2) a cassette containing the partial HIS3 marker, his3Δ3′, situated next to an HO endonuclease restriction site, and 3) flanking yeast DNA sequence homologous to the intended site of integration in the yeast genome. The desired recombinant plasmid product was initially produced via standard cloning methods and its sequence confirmed by PCR screens and DNA sequencing, but it contained several mutations. We corrected these by site‐directed mutagenesis and confirmed the corrected sequence by DNA sequencing. Additionally, during site‐directed mutagenesis one of the integrated flanking sequences in Plasmid 1 was lost, but was reintroduced via restriction digest and ligation. The second plasmid is designed to remove an unwanted HO endonuclease site present in the target yeast strain by replacing this portion of the yeast chromosome with DNA sequence lacking the HO endonuclease cassette. Making Plasmid 2 entailed PCR amplification of several DNA fragments which were then linked to each other by adaptamer‐mediated PCR. The resulting linked PCR product was ligated into the pGEMT‐Easy vector and E. coli transformants were screened. Plasmids 1 and 2 will enable creation of the specialized yeast strain for fluorescence microscopy to elucidate the timing of the end‐processing steps in SSA.Support or Funding InformationThe National Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.