Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2002 Dec;66(4):630-70, table of contents.
doi: 10.1128/MMBR.66.4.630-670.2002.

Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair

Lorraine S Symington  1
Affiliations
Review

Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair

Lorraine S Symington. Microbiol Mol Biol Rev. 2002 Dec.

Abstract

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Models for the repair of DSBs. (A) In the DSBR model, the ends are processed to yield 3′ single-stranded tails. The 3′ ends invade the homologous duplex, priming DNA synthesis. After ligation, a dHJ intermediate is formed, which can subsequently be resolved by endonucleolytic cleavage of the two Holliday junctions to generate crossover or noncrossover products. (B) In the SDSA model, the ends are processed to yield 3′ single-stranded tails, one of which invades the homologous duplex, priming DNA synthesis. The displacement loop (D-loop) formed by strand invasion could be extended by DNA synthesis or could migrate with the newly synthesized DNA. After displacement from the donor duplex, the nascent strand pairs with the other 3′ single-stranded tail and DNA synthesis completes repair. (C) The initial steps in the BIR model are the same as in the SDSA model, but DNA synthesis from the invading strand continues to the end of the DNA molecule. (D) In the SSA model, a DSB made between direct repeats is subject to resection to generate 3′ single-stranded tails. When complementary sequences are revealed due to extensive resection, the single-stranded DNA anneals, resulting in deletion of one of the repeats and the intervening DNA. The 3′ tails are endonucleolytically removed, and the nicks are ligated. The 3′ ends are indicated by arrowheads.
FIG. 2.
FIG. 2.
Mitotic recombination in diploids. A diploid containing leu2 heteroalleles and heterozygous for HIS4 is shown. Gene conversion associated with crossing over gives rise to Leu+ His+ or Leu+ His− recombinants, whereas gene conversion events maintain heterozygosity at HIS4. A BIR event cannot be distinguished from a G2 crossover unless the reciprocal product is recovered.
FIG. 3.
FIG. 3.
Use of a direct-repeat substrate to measure mitotic recombination. The substrate contains leu2 heteroalleles separated by a copy of the URA3 gene. Leu+ Ura+ recombinants can be generated by intrachromatid or sister chromatid conversion or by unequal sister chromatid exchange. Ura− recombination events can occur by an intrachromatid or unequal sister chromatid crossover, by SSA, or by replication slip mispairing.
FIG. 4.
FIG. 4.
Inverted-repeat substrates. (A) The ade2 heteroalleles can recombine to Ade+ by conversion or inversion (apparent crossover) of the intervening DNA. (B) A long-tract conversion event between sister chromatids can result in an Ade+ product with the intervening DNA inverted.
FIG. 5.
FIG. 5.
Mating-type switching. (A) Cartoon of chromosome III showing the silent cassettes, HMLα and HMRa and the expressed MATα locus. HO endonuclease cleaves between the Y and Z sequences, and repair using the HMRa donor results in switching to MATa. Regions of homology flanking Yα and Ya are indicated by W, X, and Z. (B) After HO cleavage, the 5′ end on the distal side of the break is resected and the resulting 3′ single-stranded tail invades the HMRa locus. DNA synthesis is primed from the invading strand, duplicating Ya sequences. Lagging-strand synthesis initiated from the D-loop results in synthesis of the other strand. The mechanism for removal of the Yα strands is currently unknown. (C) Schematic representation of a Southern blot showing the kinetics of mating-type switching. Switching to Ya results in transfer of a novel StyI site and therefore can be monitored by Southern blot analysis of DNA extracted after HO induction and digested with StyI. Resection of the 5′ strands beyond the distal StyI site results in resistance to digestion to StyI because the site is within ssDNA.
FIG. 6.
FIG. 6.
Gene targeting. (A) When yeast cells are transformed with a replicating plasmid (ARS plasmid) containing a double-strand break or gap within a region of the plasmid with homology to the yeast genome, the break or gap is repaired by gene conversion from the chromosomal locus. The plasmid remains episomal if gene conversion without an associated crossover occurs but is integrated into the genome if conversion is associated with crossing over. If the plasmid contains a CEN sequence, only conversion events can give rise to viable transformants. If the plasmid contains no origin of replication (ARS), only integration events are observed. (B) Ends-out gene targeting refers to replacement of chromosomal sequences with sequences present on a linear DNA fragment introduced into cells by transformation. Gene targeting is thought to occur by invasion of the two ends into the chromosomal locus followed by resolution of the resulting Holliday junctions. Extensive DNA synthesis could be primed from the invading 3′ ends prior to Holliday junction resolution, or resolution could occur by replication to the end of the chromosome. Gene targeting could also result from integration of a single strand of the targeting fragment followed by trimming the D-loop and mismatch repair.
FIG. 7.
FIG. 7.
Physical assays for meiotic recombination. (A) The hot spot created by insertion of the LEU2 gene at HIS4 in the SK1 strain background is shown. The end of the LEU2 gene contains a site for meiosis-specific DSBs flanked by restriction sites that can be used to distinguish between DSB fragments, and the two parental and two recombinant fragments. The white and black boxes indicate insertions of heterologous sequences used to distinguish the parental molecules by using strand-specific probes. (B) Three types of joint molecules are expected: Mom-Mom intersister joint molecules, Dad-Dad intersister joint molecules, and Mom-Dad interhomologue joint molecules. Size and hybridization to strand specific probes distinguish the three types of joint molecules. (C) Cartoon of a neutral-neutral two-dimensional gel showing separation of joint molecules from the parental and recombinant fragments. The interhomologue joint molecules are more abundant than the intersister joint molecules in wild-type cells. (Adapted from Fig. 1 of reference with permission)
FIG. 8.
FIG. 8.
Schematic representation of Mre11, Rad50, and Xrs2 (Nbs1). The phosphodiesterase motifs of Mre11 are labeled MI through MIV, and DNA binding sites are labeled DB site A and B. The Mre11-D16A, Mre11-D56N, Mre11-H125N, Mre11-H213Y, and Mre11-6 mutants are nuclease defective in vitro. Residue Pro84 is mutated in the mre11-S allele, and Pro162 is mutated in the mre11-1 temperature-sensitive allele. The mre11-N113S and mre11-Q623Z alleles correspond to the mutations in the A-TLD patients. Rad50 contains two coiled-coil domains separating the Walker A and B motifs for NTP binding and hydrolysis. The hook domain, containing the conserved CXXC motif, is located between the two coiled-coil domains. The positions of the rad50S alleles, rad50-R20M and rad50-K81I, are shown.
FIG. 9.
FIG. 9.
Models for the Rad50-Mre11 complex. A dimer of Rad50 could form by antiparallel intermolecular interaction to position the Walker A motif from one monomer next to the Walker B motif of the other. A dimer of Mre11 binds adjacent to the head-tail region of Rad50. Recent results are more consistent with an intramolecular interaction between the coiled-coil domains of Rad50, with the dimer held together by a dimer of Mre11. Interactions between the hinge domains could connect two dimers of Rad50 and two dimers of Mre11. The length of the Rad50 tetramer is consistent with the distance between sister chromatids in eukaryotes. The Mre11 and Rad50 head-tail domains are envisioned to interact with DNA.
FIG. 10.
FIG. 10.
Models for DSB processing by the MRX complex and Exo1. In wild-type cells, ends are processed by unwinding of ends and endonucleolytic cleavage of the 5′ strand by the Mre11 nuclease. Unwinding could be mediated by the weak unwinding activity of the Mre11 complex or by association with a DNA helicase. In the absence of the MRX complex, Exo1 inefficiently processes the ends. The M*RX complex in cells expressing the mre11-H125N allele is still able to unwind ends, and other nucleases remove the 5′ single-stranded tails.
FIG. 11.
FIG. 11.
Telomere maintenance by recombination in the absence of telomerase. In the absence of telomerase, telomeres become progressively shorter and are maintained by RAD52-dependent recombination. Two types of recombination events give rise to survivors: RAD51-, RAD54-, and RAD57-dependent recombination between Y′ sequences, and RAD50- and RAD59-dependent recombination between T G1-3 tracts. Y′ elements are marked by open boxes, and tracts of TG1-3 are indicated by shaded boxes.
FIG. 12.
FIG. 12.
Substrates used for in vitro strand exchange assays. (A) Pairing of a circular single-stranded molecule with linear duplex results in the formation of a nicked circular duplex product and a displaced linear single strand. (B) Invasion of a linear single-stranded molecule into supercoiled circular DNA results in the formation of a joint molecule with a D-loop. (C) Strand exchange between an unlabeled single-stranded oligonucleotide and labeled double-stranded oligonucleotides is detected by displacement of a labeled single-stranded oligonucleotide.
FIG. 13.
FIG. 13.
Role of the Rad1-Rad10 nuclease in recombination. (A) In the SSA reaction, resected 3′ single-stranded tails are first coated with RPA; the RPA is then displaced by Rad52 to promote annealing of the complementary single strands. After annealing, 3′ heterologous tails are removed by the Rad1-Rad10 endonuclease, which cleaves at the junction between dsDNA and ssDNA. Gaps are filled by repair synthesis and ligated. (B) If the invading 3′ end is heterologous to the donor, then the 3′ end cannot be used to template DNA synthesis. The branched structure is cleaved by Rad1-Rad10, creating a primer for DNA synthesis. The second 3′ heterologous tail is also removed by Rad1-Rad10 for the second round of DNA synthesis predicted by the SDSA model (shown) or DSBR model.
FIG. 14.
FIG. 14.
The BIR/SSA model for repair of DSBs within inverted repeat plasmids. After formation of the DSB, ends are processed and one end invades the other repeat (drawn here as an intramolecular event, but it could also occur intermolecularly). If DNA synthesis extends to the end of the molecule, small repeats will be present at both ends of the linear molecule. Following resection of the ends, the repeats at either end of the molecule can pair with an internal repeat by SSA, generating either noncrossover or apparent crossover products.
FIG. 15.
FIG. 15.
Model for strand invasion by Rad51 and the mediator proteins, Rad52, Rad54, and Rad55-Rad57. Single-stranded tails produced at break sites are coated by RPA. Rad52 interacts with RPA targeting Rad51 to the ssDNA. Rad52 is thought to displace RPA and promote the binding of Rad51 to ssDNA. This initial interaction between Rad51 and ssDNA is thought to be stabilized by Rad55-Rad57 to allow cooperative binding by Rad51. Rad54 interacts with the Rad51 nucleoprotein filament and promotes the unwinding of duplex DNA for pairing between the donor DNA and the incoming single strand.
FIG. 16.
FIG. 16.
Role of recombination in restoring collapsed or regressed replication forks. The replication fork collapses if it encounters a nick on the template strand. After ligation of the lagging strand to the template, the broken arm invades, forming a D-loop. Replication can then be primed from the invading strand. Resolution of the Holliday junction (HJ) restores the replication fork. A replication-blocking lesion stalls replication, and the fork regresses by pairing of the nascent strands. If the lagging strand has progressed ahead of the leading strand, the single-strand extension on the lagging strand can serve as a primer for synthesis of the leading strand. If the Holliday junction is cleaved, an end is made for strand invasion, as shown in the left panel. Alternatively, branch migration of the regressed fork can restore the replication fork.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aboussekhra, A., R. Chanet, A. Adjiri, and F. Fabre. 1992. Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins. Mol. Cell. Biol. 12:3224-3234. - PMC - PubMed
    1. Adair, G. M., R. L. Rolig, D. Moore-Faver, M. Zabelshansky, J. H. Wilson, and R. S. Nairn. 2000. Role of ERCC1 in removal of long non-homologous tails during targeted homologous recombination. EMBO J. 19:5552-5561. - PMC - PubMed
    1. Adzuma, K., T. Ogawa, and H. Ogawa. 1984. Primary structure of the RAD52 gene in Saccharomyces cerevisiae. Mol. Cell. Biol. 4:2735-2744. - PMC - PubMed
    1. Aguilera, A. 1995. Genetic evidence for different RAD52-dependent intrachromosomal recombination pathways in Saccharomyces cerevisiae. Curr. Genet. 27:298-305. - PubMed
    1. Aguilera, A., and H. L. Klein. 1989. Yeast intrachromosomal recombination: long gene conversion tracts are preferentially associated with reciprocal exchange and require the RAD1 and RAD3 gene products. Genetics 123:683-694. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources