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. 2007 Dec;3(12):e228.
doi: 10.1371/journal.pgen.0030228.

Genome-wide analysis of Rad52 foci reveals diverse mechanisms impacting recombination

David Alvaro  1 , Michael Lisby, Rodney Rothstein
Affiliations

Genome-wide analysis of Rad52 foci reveals diverse mechanisms impacting recombination

David Alvaro et al. PLoS Genet. 2007 Dec.

Abstract

To investigate the DNA damage response, we undertook a genome-wide study in Saccharomyces cerevisiae and identified 86 gene deletions that lead to increased levels of spontaneous Rad52 foci in proliferating diploid cells. More than half of the genes are conserved across species ranging from yeast to humans. Along with genes involved in DNA replication, repair, and chromatin remodeling, we found 22 previously uncharacterized open reading frames. Analysis of recombination rates and synthetic genetic interactions with rad52Delta suggests that multiple mechanisms are responsible for elevated levels of spontaneous Rad52 foci, including increased production of recombinogenic lesions, sister chromatid recombination defects, and improper focus assembly/disassembly. Our cell biological approach demonstrates the diversity of processes that converge on homologous recombination, protect against spontaneous DNA damage, and facilitate efficient repair.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Rad52-YFP Focus Screen Using Systematic Hybrid LOH
(A) Method for preparation of hybrid LOH strains for screening, permitting simultaneous complementation of recessive factors in the library strains and introduction of the Rad52-YFP plasmid through mating [12]. KanMX-marked gene deletion strains are mated with corresponding conditional centromere strains. Growth in galactose medium drives transcription through the centromere destabilizing the chromosome and 5-FOA selects for cells that have lost the conditional chromosome. In most cases, loss is followed by endoduplication of the monosomic chromosome, generating a 2n hybrid diploid homozygous for the gene deletion. (B) Spontaneous Rad52-YFP foci in a wild-type strain and in an irc6Δ deletion, a strain identified in the screen with an increased fraction of cells containing spontaneous foci.
Figure 2
Figure 2. Distribution of 4,805 Library Gene Deletions for Rad52-YFP Focus Levels
The levels are the percentage of cells with one or more spontaneous Rad52-YFP foci among 200–400 homozygous hybrid diploid cells screened. The strains were divided into four subsets based upon focus levels: (A) zero foci (4.8% of the library); (B) 0.1%–15% foci (90% of the library); (C) 15%–20% foci (3.0% of the library); and (D) >20% foci (2.2% of the library). The inset represents the distribution of 354 repetitions of the wild-type parent strain BY4742 scored concurrently with the gene deletions during the screen.
Figure 3
Figure 3. Recombination Assays Used in This Study
(A) The direct repeat recombination assay measures intra-chromosomal and sister chromatid recombination events that generate a functional LEU2 allele from two leu2 heteroalleles. The URA3 marker between the heteroalleles permits discrimination between sister chromatid conversion events (Leu+ Ura+ recombinants) and SSA (Leu+ Ura− recombinants). (B) The interhomolog recombination assay measures recombination between leu2 heteroalleles on homologous chromosomes in diploid cells. Chromosomes are shown following DNA synthesis and reflect a pair of sister chromatids for each chromosome homolog. A recombinogenic lesion occurring at the LEU2 locus is most often repaired using the template on the sister chromatid, which is a conservative event resulting in no loss or gain of genetic information (left). However, a gene conversion event or reciprocal exchange between two homologous chromosomes can generate a functional LEU2 allele (right).
Figure 4
Figure 4. Pathways Leading to Increased Rad52 Foci Are Revealed by This Study
Class I mutants block HR subsequent to the formation of Rad52 foci. Class II mutants stimulate the formation of DNA lesions, which is reflected as increased focus formation and results in increased sister chromatid and interhomolog recombination. Class III mutants decrease the efficiency of sister chromatid recombination through effects on cohesion, chromatin architecture, HR, and other mechanisms, thereby increasing the duration of Rad52 foci (indicated by the meandering line). Class IV mutants do not have a global effect on homologous recombination. However, the focus phenotype in some mutants reflects recombination at specific sites such as rDNA or telomeres. Other mutants may cause the division of cells prior to resolution of HR foci, or lead to the generation of lesions that cannot be repaired, resulting in lethality. For all classes, mutations with an asterisk and highlighted in yellow, sensitize cells to the absence of Rad52 (see Table 2).
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References

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