Two Different Types of Double-Strand Breaks in Saccharomyces cerevisiae Are Repaired by Similar RAD52-Independent, Nonhomologous Recombination Events

Abstract

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells.

High-resolution profiling of γH2AX around DNA double strand breaks in the mammalian genome

“…Genotoxic drugs and radiation, often used to generate DSBs, induce random breaks throughout the genome (thus heterogeneously across the cell population), which are inappropriate for subsequent ChIP analyses. A few systems have been developed to induce and study localized DSBs in yeast and mammalian cells (Kramer et al , 1994; Rouet et al , 1994; Wolner et al , 2003; Berkovich et al , 2007; Savic et al , 2009). However, none of them permits precise analysis of γH2AX spreading around many breaks located in various chromatin contexts.…”

“…However, none of them permits precise analysis of γH2AX spreading around many breaks located in various chromatin contexts. On one hand, the I‐SceI‐based (Rouet et al , 1994) and the HO‐mediated (Kramer et al , 1994) DSB‐inducible systems, as well as the VDJ locus (Savic et al , 2009), lead to only one localized DSB, generating insufficient γH2AX coverage to perform statistical analyses. On the other hand, the I‐PpoI‐based DSB‐inducible system (Berkovich et al , 2007) generates several endogenous sequence‐specific DSBs.…”

Rapid, scalable, combinatorial genome engineering by marker-less enrichment and recombination of genetically engineered loci in yeast

“…CRISPR-Cas9 enables the precise and marker-less editing of both essential and non-essential yeast loci owing to significantly lower error-prone non-homologous end joining (NHEJ) in yeast relative to HDR. 25 Therefore, Cas9-single guide RNA (sgRNA)-induced lethality serves as a rapid test for a functional CRISPR plasmid (pCas9-sgRNA locus ) in yeast (colony-forming units observed sensitive [CFU OS ]) compared with the Cas9 alone (CFUs expected [CFU E ]; CFU OS /CFU E = ∼0). In contrast, a yeast strain that harbors a corresponding engineered locus resistant to further targeting allows the survival of colonies (CFUs observed resistant [CFU OR ]) similar to the pCas9 alone (CFU OR /CFU E = ∼1) ( Figure 1 A).…”

Protocol to measure end resection intermediates at sequence-specific DNA double-strand breaks by quantitative polymerase chain reaction using ER-AsiSI U2OS cells

“…Researchers also used different sequence-specific DSB-inducible systems such as Fok1, A si SI, I-PpoI, I-SceI-based and HO-dependent systems to induce with a high degree of precision DSBs at specific genomic loci in human and yeast cells. 5 , 6 , 7 , 8 , 9 These systems allowed them to study DSB response while ensuring no break occurs elsewhere in the genome. 5 , 6 , 7 , 8 , 9 Another tool is Zinc-finger nucleases (ZFNs), which were built by attaching zinc-finger DNA-binding domains to the catalytic domain of the Fok I endonuclease.…”

“… 5 , 6 , 7 , 8 , 9 These systems allowed them to study DSB response while ensuring no break occurs elsewhere in the genome. 5 , 6 , 7 , 8 , 9 Another tool is Zinc-finger nucleases (ZFNs), which were built by attaching zinc-finger DNA-binding domains to the catalytic domain of the Fok I endonuclease. 10 While ZFNs were used for targeted gene editing in the eukaryotes, 11 they were toxic and had a high frequency of off-target mutations.…”