| The repair of double-strand breaks (DSBs) by homologous recombination requires new DNA synthesis, but this essential step has not been well-characterized. The goal of this thesis was to analyze intermediates of mitotic DSB repair at the mating type locus (MAT) in Saccharomyces cerevisiae , using mutant strains thermosensitive for essential replication factors. Analysis of both leading strand polymerases indicates that DNA polymerase ϵ is the major repair polymerase involved, but is partially substituted by DNA polymerase δ. The processivity cofactors PCNA and RFC are essential, even to synthesize as little as 30 nucleotides following strand invasion. Surprisingly, mutants of the lagging strand machinery, DNA polymerase α (pol1-17), DNA primase (pri2-1) and Rad27 (rad27Δ), also greatly inhibit intermediate steps of DNA synthesis and final product formation. These results argue against the classic Szostak et al. (1983) model for DSB repair, in which only leading strand repair synthesis is presumed. We propose a new model for DSB repair, in which a strand invasion creates a modified replication fork, involving leading and lagging strand synthesis from the donor template.; When a DSB at the MAT locus is made unrepairable by deleting the homologous donors, cells normally arrest in G2/M, but then “adapt” to the DNA damage and continue growing, until they eventually die from extensive chromosome loss. The 5′ to 3′ ′ exonuclease activities of several DNA repair proteins involved in nonhomologous endjoining, including Mre11p, Rad50p, and Hdf1p were monitored. We have shown that a deletion of the yeast Ku70p homolog, Hdf1p, shows a two-fold increase in degradation and prevents the cell from escaping G2 arrest. The permanent G2 arrest depends on the RAD9 and RAD17 checkpoint genes. This arrest is suppressed in mre11 and rad50 mutant strains, which slow down degradation, as well as in a mutant of the single-stranded DNA (ssDNA) binding protein, RPA. Therefore, this adaptation process is exquisitively sensitive to the amount of 3′ ended ssDNA tails produced upon DSB formation, and depends on the recognition of the ssDNA by RPA. |
