Open AccessEnhancement of Saccharomyces cerevisiae End-Joining Efficiency by Cell Growth Stage but Not by Impairment of Recombination
Author(s) -
Elissa Karathanasis,
Thomas E. Wilson
Publication year - 2002
Publication title -
genetics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.792
H-Index - 246
eISSN - 1943-2631
pISSN - 0016-6731
DOI - 10.1093/genetics/161.3.1015
Subject(s) - non homologous end joining , biology , homologous recombination , endonuclease , saccharomyces cerevisiae , genetics , plasmid , dna repair , cell cycle , dna , gene conversion , homology directed repair , flp frt recombination , gene , microbiology and biotechnology , genome , recombination , dna mismatch repair , genetic recombination
Cells can repair DNA double-strand breaks by both homologous and nonhomologous mechanisms. To explore the basis of pathway utilization, we developed both plasmid and chromosomal yeast repair assays in which breaks are created with restriction endonucleases so that nonhomologous end-joining (NHEJ) competes with the single-strand annealing (SSA) recombination pathway, which we show acts with high efficiency via terminal direct repeats of only 28 bp and with reduced but measurable efficiency at 10 bp. The chromosomal assay utilizes a novel approach termed suicide deletion in which the endonuclease cleaves its own gene from the chromosome, thereby ending the futile cleavage cycle that otherwise prevents detection of simple-religation events. Eliminating SSA as a possibility in either assay, either by removal of the direct repeat or by mutation of RAD52, increased the relative but not the absolute efficiency of NHEJ. In contrast, the apparent efficiency of NHEJ was specifically increased in the G1 stage of the haploid cell cycle, as well as by the glucose depletion-signaled transition to stationary phase. The combined results argue against a model in which pathway utilization is determined by a passive competition. Instead, they demonstrate an active regulation designed to optimize the likelihood of genome restoration based on cell state.