Homologous recombination (HR), a repair mechanism that depends on DNA sequence homology, underlies a number of important DNA processes that act to both stabilize and diversify a genome. In mitotic cells, HR functions to maintain the integrity of the genome through such processes as the repair of DNA double-strand breaks (DSBs), the maintenance of rDNA copy number, and the rescue of collapsed replication forks. HR is entirely conservative when it occurs following DNA replication where a sister chromatid is available as a template. However, utilization of sequences on a homologous chromosome can lead to crossovers and potential loss of heterozygosity (LOH), while recombination at ectopic or repeated sequences may lead to genomic rearrangements such as deletions, duplications, and translocations (reviewed in [1
In Saccharomyces cerevisiae
, Rad52 is the defining member of an epistasis group that includes: RecA homologs Rad51, Rad55, Rad57, and Dmc1; putative SWI/SNF family ATPase Rad54; Rad52 homolog Rad59 and Mre11, Xrs2, and Rad50. The Rad52 epistasis group is essential for HR. Rad52 binds single-stranded DNA in vitro and has been shown to stimulate DNA annealing and to enhance Rad51-catalyzed strand invasion [2
In response to DNA damage, proteins involved in HR relocalize into discrete subnuclear foci. Fluorescently tagged repair and checkpoint proteins have been used to explore the composition and dynamics of these foci, which are giga-dalton-sized assemblies of proteins [6
]. Repair foci colocalize with fluorescently tagged inducible DSB sites, regions of single-stranded DNA, and sites of unscheduled DNA synthesis [7
]. Multiple DSBs often colocalize at a single focus showing that foci reflect recombination centers capable of the simultaneous repair of more than one lesion. The assembly of proteins into repair foci is a coordinated process beginning with detection of damage by the Mre11/Rad50/Xrs2 complex. Next, checkpoint proteins are bound and activated to arrest cell cycle progression until completion of repair. The lesion is repaired through HR performed by the Rad52 epistasis group proteins and finally the repair apparatus is disassembled [10
]. From a cell biology perspective, Rad52 focus formation is an excellent marker for HR, since it is required for the recruitment of all other HR proteins into repair foci.
While exogenous DNA damage greatly stimulates the formation of Rad52 foci, foci also form spontaneously in S phase cells, likely reflecting the repair of spontaneous DNA lesions such as DSBs, nicks, and single-stranded gaps [6
]. Time-lapse microscopy indicates that foci form in approximately 50% of cells during S phase and most spontaneous foci persist for less than 10 min [7
]. Since spontaneous foci generally last for only a fraction of S phase, they are observed in 20% of S phase cells in a population of logarithmically growing cells (5% of the total population). Mutants defective in various aspects of DNA metabolism, including damage checkpoints (mec1 sml1
), HR (rad51
Δ), and DNA replication (pol12–100
) exhibit elevated levels of spontaneous foci [6
]. This elevation may be the consequence of an increased incidence of focus formation reflecting the generation of more DNA lesions, or the consequence of foci that persist over time resulting from an alteration in the dynamics of focus assembly/disassembly.
Here we report the results of a genome-wide screen designed to identify gene deletions that significantly alter levels of spontaneous Rad52 foci. The set of gene deletions identified includes many known genes involved in DNA and chromatin processes such as replication, repair, silencing, and chromosome segregation, as well as a number of other processes with no reported link to HR. In addition, 22 previously uncharacterized ORFs designated IRC2–11, 13–16, 18–25 (Increased Recombination Centers) were identified. Measurement of HR between sister chromatids and between homologous chromosomes in these focus mutants established four different classes demonstrating that several distinct mechanisms are involved in precipitating increased Rad52 foci. Furthermore, several IRC genes exhibit synthetic interactions with a rad52Δ allele suggesting a direct role in the maintenance of genomic integrity.