Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

DNA repair is critical for genome stability and tumor suppression in higher eukaryotes. DNA double-strand breaks (DSBs) are critical lesions that result from exposure to exogenous agents (ionizing radiation or genotoxic chemicals) and from endogenous sources, such as nucleases and spontaneous DNA lesions that cause replication fork collapse. DSBs can be repaired by homologous recombination (HR) or by nonhomologous end joining (NHEJ). In the yeast Saccharomyces cerevisiae, the primary DSB repair pathway is homologous recombination (HR), and the key HR proteins are members of the Rad52 epistasis group (Rad51, Rad52, Rad54, Rad55, Rad57, and Rad59). The Mre11/Rad50/Xrs2 (MRX) complex has roles in both HR and NHEJ. The most common HR outcome is gene conversion, a conservative and highly accurate mode of DSB repair that occurs with or without an associated crossover. In cells with mild to severe HR defects, other outcomes may occur, including chromosome loss or nonconservative repair by either break-induced replication (BIR) or single-strand annealing (SSA) (10, 16, 25, 27). Inefficient or inaccurate DSB repair by either HR or NHEJ has been linked to the development and progression of cancer (28, 59). DSB repair by HR involves a series of steps that begins with 5′-to-3′ single-strand resection regulated by MRX, and binding of the single-stranded DNA (ssDNA) tail by replication protein A. Replication protein A is then exchanged with Rad51 to produce Rad51 nucleoprotein filaments that are capable of searching for, and invading, homologous sequences elsewhere in the genome (sister chromatids, homologous chromosomes, or repeated sequences). The Rad51-related protein Dmc1 is also required for meiotic HR (32). Rad51 filament formation is promoted by the “mediator” proteins Rad52, Rad55, and Rad57; Rad54 also mediates Rad51 filament formation in vitro, although filaments form normally in rad54Δ mutants in vivo (19, 51, 54, 60). Genetic and biochemical studies suggest that Rad54 promotes invasion of the intact (donor) duplex by the Rad51 filament and branch migration of the joint molecule producing heteroduplex DNA (hDNA) and dissociates Rad51 from ssDNA to allow repair synthesis by extension of the invading 3′ end (4, 15, 40, 47, 48). The invading 3′ end is extended by repair synthesis past the site of the DSB and can then dissociate from the donor duplex and anneal with the other ssDNA tail in a process termed synthesis-dependent strand annealing, a noncrossover pathway. Alternatively, both ends may invade a donor, producing Holliday junction intermediates that can be resolved with or without crossovers. Mismatches arise in hDNA when interacting duplexes have sequence differences (“markers”). Mismatch repair is typically biased, with the strand from the donor (unbroken) duplex used as a mismatch repair template, resulting in regions of marker loss in the recipient (broken) duplex that are termed conversion tracts (38). Because most mismatch repair proceeds by a long-patch, excision-based mechanism, conversion tracts are usually continuous, i.e., when two markers flanking a central marker are converted, the central marker almost always coconverts. Thus, conversion tracts result from mismatch repair of hDNA produced during strand invasion and branch migration and when an extended 3′ end anneals to ssDNA during synthesis-dependent strand annealing. The Rad51 filament plays a central role in strand invasion, and DSB repair via gene conversion is severely reduced or eliminated in the rad51Δ mutant, with repair instead occurring by SSA or BIR. With only a single DSB, SSA is limited to interactions between linked, direct repeats as resection to ssDNA tails exposes complementary single strands that anneal to repair the break. SSA may be important for repair when two or more DSBs occur near each other (49). Because SSA does not require strand invasion, it is independent of Rad51, Rad54, Rad55, and Rad57 but requires the strand-annealing activity of Rad52 (10, 27, 49). BIR was initially described in rad51Δ cells (25, 46). Several studies have revealed an inefficient Rad51-independent BIR mechanism that requires Rad59, Tid1, and MRX and a more efficient Rad51-dependent mechanism that requires Rad52, Rad55, Rad57, Rad54, and Ku (6, 16, 26, 46). Even the more efficient BIR mechanism is rare or absent in wild-type cells when gene conversion is possible (25, 35, 46), although BIR appears to be important for telomerase-independent telomere maintenance (24). Despite the central role of Rad51 in strand invasion, Rad51-independent gene conversion has been detected in certain contexts. Spontaneous gene conversion between inverted repeats is reduced only 4-fold by deletion of RAD51, whereas deletion of RAD52 reduces these events by 3,000-fold (42). Ectopic gene conversion involving repair of plasmid double-strand gaps with plasmid or chromosomal repair templates was readily detected in the rad51Δ mutant, albeit ∼100-fold less efficiently than in the wild type; these recombination events were dependent on Rad52 and Rad59 (2). Yeast mating-type switching involves intrachromosomal gene conversion resulting from repair of an HO nuclease-induced DSB at the MAT locus. This is a lethal event in the rad51Δ mutant; however, MAT switching does occur in the rad51Δ mutant when interacting alleles are present on plasmids. These plasmid events were originally explained as reflecting a relaxed requirement for Rad51 when donor sequences are not silenced in heterochromatin, as in normal MAT switching (50), but an alternative explanation is that the plasmid events depend on BIR between closely linked alleles (2). Thus, Rad51-independent DSB repair by gene conversion has been detected only for ectopic events (inverted repeats, or plasmid-chromosome or plasmid-plasmid interactions); there are no prior reports of such repair between chromosomal (allelic) loci in the absence of Rad51 (17). By using a sensitive DSB repair assay, we demonstrate that yeast is capable of Rad51-independent DSB repair resulting in interchromosomal gene conversion. The efficiency of this repair is similar to that of Rad51-independent BIR, and these processes share similar genetic requirements: both are independent of Rad55, Rad57, and Dmc1 but require Rad52. Spontaneous gene conversion, however, was detected in the absence of Rad51 or Rad52, but not both proteins. Crossovers associated with conversion occur at about the same frequency during Rad51-dependent and -independent events, but Rad51-independent gene conversion tracts were frequently discontinuous and generally longer than in the wild type, suggesting that hDNA forms or is processed differently in the absence of Rad51.