Targeted Inactivation of Mouse RAD52 Reduces Homologous Recombination but Not Resistance to Ionizing Radiation (original) (raw)
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Homologous recombination, but not DNA repair, is reduced in vertebrate cells deficient in RAD52
Molecular and cellular biology, 1998
Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generated RAD52(-/-) mutants of the chicken B-cell line DT40. Surprisingly, RAD52(-/-) cells were not hypersensitive to DNA damages induced by gamma-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52(-/-) cells as in wild-type cells. Targeted integration frequencies, however, were consistently r...
Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair
Microbiology and Molecular Biology Reviews, 2002
SUMMARY 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 fo...
Mutation Research/DNA Repair, 1997
The yeast Saccharomyces cereÕisiae RAD52 gene is involved in recombination and DNA double-strand break repair. Recently, mouse and human homologs of the yeast RAD52 gene have been identified. Here we present the genomic organization of the mouse RAD52 gene. It consists of 12 exons ranging in size from 67 to 374 bp spread over a region of approximately 18 kb. The first ATG is located in exon 2. Analysis of the promoter region revealed no classical promoter elements such as CCAAT or TATA boxes. Transcriptional mapping analysis revealed one major transcription start point. Analogous to the situation in yeast, transcription of the RAD52 gene in human skin fibroblasts and mouse L tk y cells was not induced by methyl methanesulfonate treatment. Furthermore, no specific alteration in human RAD52 expression levels throughout the cell cycle was observed.
Nucleic acids research, 2017
RAD52 is a homologous recombination (HR) protein that is conserved from bacteriophage to humans. Simultaneously attenuating expression of both the RAD52 gene, and the HR and tumor suppressor gene, BRCA2, in human cells synergistically reduces HR - indicating that RAD52 and BRCA2 control independent mechanisms of HR. We have expressed the human RAD52 gene (HsRAD52) in budding yeast strains lacking the endogenous RAD52 gene and found that HsRAD52 supports repair of DNA double-strand breaks (DSB) by a mechanism of HR that conserves genome structure. Importantly, this mechanism of HR is independent of RAD51, which encodes the central strand exchange protein in yeast required for conservative HR. In contrast, BRCA2 exerts its effect on HR in human cells together with HsRAD51, potentially explaining the synergistic effect of attenuating the expression of both HsRAD52 and BRCA2. This suggests that multiple mechanisms of conservative DSB repair may contribute to tumor suppression in human c...
Recruitment of the Recombinational Repair Machinery to a DNA Double-Strand Break in Yeast
Molecular Cell, 2003
viewed in Pâ ques and Haber, 1999; Sung et al., 2000). In the mouse, a homozygous null allele of RAD51 leads to embryonic lethality (Tsuzuki et al., 1996), and muta-Program in Molecular Medicine tions in RAD genes are associated with a spectrum of University of Massachusetts Medical School diseases, including cancer (reviewed in Ivanov and Ha-Worcester, Massachusetts 01605 ber, 1997; Jasin, 2000; Michelson and Weinert, 2000). 2 Institute of Biotechnology and Studies in yeast have suggested a sequence of molec-Department of Molecular Medicine ular events that occur following formation of a DSB (re-. First, the 5Ј ends of DNA that flank San Antonio, Texas 78245 the break are resected by an exonuclease. Rad51p, a functional homolog of the E. coli RecA recombinase, then binds the exposed single-stranded tails forming a right-Summary handed helical nucleoprotein filament. In vitro, Rad52p (Sung, 1997a) and a Rad55p/Rad57p heterodimer (Sung, Repair of DNA double-strand breaks (DSBs) by homol-1997b) can promote this early step by overcoming the ogous recombination requires members of the RAD52 inhibitory effects of the heterotrimeric single-stranded epistasis group. Here we use chromatin immunopre-DNA binding protein, RPA. The Rad51p nucleoprotein cipitation (ChIP) to examine the temporal order of filament is then believed to function in cooperation with recruitment of Rad51p, Rad52p, Rad54p, Rad55p, Rad54p to search the genome for a homologous pairing and RPA to a single, induced DSB in yeast. Our results partner and to form a heteroduplex "joint molecule" (Petsuggest a sequential, interdependent assembly of ukhova et al., 1998, 2000). Joint molecule formation is Rad proteins adjacent to the DSB initiated by binding followed by extension of the incoming strand by DNA of Rad51p. ChIP time courses from various mutant polymerases and branch migration, ultimately leading strains and additional biochemical studies suggest to restoration of the genetic information spanning the that Rad52p, Rad55p, and Rad54p each help promote break (reviewed in Pâ ques and Haber, 1999). the formation and/or stabilization of the Rad51p nu-Much less is known about how Rad proteins functioncleoprotein filament. We also find that all four Rad ally cooperate during DSB repair in vivo. Immunofluoresproteins associate with homologous donor sequences cence studies have shown that Rad51p, Rad52p, and during strand invasion. These studies provide a near Rad54p colocalize to "foci" in response to DNA damage comprehensive view of the molecular events required in vivo (Haaf et al., 1995; Tan et al., 1999), suggesting for the in vivo assembly of a functional Rad51p presynthat Rad proteins might function together within a larger, aptic filament. multiprotein complex. Consistent with this view, coimmunoprecipitation and yeast two-hybrid assays have Introduction shown that many members of the RAD52 group can interact with each other (Golub et al., 1997; Hays et al., DNA double-strand breaks (DSBs) arise in DNA due to 1995; Johnson and Symington, 1995; Krejci et al., 2001). environmental insults such as ionizing radiation or In contrast, recent studies indicate that the composition chemical exposure. DSBs also play an important role as of the damage-induced foci are dynamic, and photointermediates in DNA replication, immunoglobulin V(D)J bleaching studies indicate that several Rad proteins recombination, meiotic and mitotic crossing-over, and have very different diffusion coefficients, suggesting that yeast mating-type switching. Failure to correctly prothey may not exist together in a preassembled protein cess these DSBs can result in deletion or insertion of complex (Essers et al., 2002). genetic information, chromosomal fragmentation, trans-We wished to dissect how Rad proteins are recruited location, and chromosome loss. and function at a DSB in vivo. Here we use chromatin Homologous recombination (HR) is a major pathway immunoprecipitation (ChIP) analyses to examine the of DSB repair in all eukaryotes and has a distinct advantemporal order of Rad protein recruitment to a single, tage over other mechanisms in that it is mostly error induced DSB in yeast. Our results suggest a sequential free. Repair of DSBs by HR requires the RAD52 epistasis pathway, where Rad51p binds first, followed by Rad52p, group, defined by the yeast RAD50, RAD51, RAD52, Rad55p, and finally Rad54p. Each of these Rad proteins RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2 genes. also associates with the homologous donor sequences These genes are highly conserved among all eukaryotes during strand invasion. We further examined the func-(Cromie et al., 2001; Pâ ques and Haber, 1999; Sung et tional interdependencies among these proteins by peral., 2000), highlighting the importance of these proteins
Genetics, 2000
Chromosomal repair was studied in stationary-phase Saccharomyces cerevisiae, including rad52/rad52 mutant strains deficient in repairing double-strand breaks (DSBs) by homologous recombination. Mutant strains suffered more chromosomal fragmentation than RAD52/RAD52 strains after treatments with cobalt-60 gamma irradiation or radiomimetic bleomycin, except after high bleomycin doses when chromosomes from rad52/rad52 strains contained fewer DSBs than chromosomes from RAD52/RAD52 strains. DNAs from both genotypes exhibited quick rejoining following gamma irradiation and sedimentation in isokinetic alkaline sucrose gradients, but only chromosomes from RAD52/RAD52 strains exhibited slower rejoining (10 min to 4 hr in growth medium). Chromosomal DSBs introduced by gamma irradiation and bleomycin were analyzed after pulsed-field gel electrophoresis. After equitoxic damage by both DNA-damaging agents, chromosomes in rad52/rad52 cells were reconstructed under nongrowth conditions [liquid hol...
Molecular and Cellular Biology, 2003
Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G 2 /M arrest. We have found that both Rad51 and Rad52 recombination proteins play key roles in adaptation. Cells lacking Rad51p fail to adapt, but deleting RAD52 suppresses rad51⌬. rad52⌬ also suppresses adaptation defects of srs2⌬ mutants but not those of yku70⌬ or tid1⌬ mutants. Neither rad54⌬ nor rad55⌬ affects adaptation. A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective. In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to singlestranded DNA (ssDNA), supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p. An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51⌬ mutants and fully restores adaptation in yku70⌬ and tid1⌬ mutants. Surprisingly, although neither rfa1-t11 nor rad52⌬ mutants are adaptation defective, the rad52⌬ rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53 after HO induction. We suggest that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p.
Cancers, 2019
Genomes are continually subjected to DNA damage whether they are induced from intrinsic physiological processes or extrinsic agents. Double-stranded breaks (DSBs) are the most injurious type of DNA damage, being induced by ionizing radiation (IR) and cytotoxic agents used in cancer treatment. The failure to repair DSBs can result in aberrant chromosomal abnormalities which lead to cancer development. An intricate network of DNA damage signaling pathways is usually activated to eliminate these damages and to restore genomic stability. These signaling pathways include the activation of cell cycle checkpoints, DNA repair mechanisms, and apoptosis induction, also known as DNA damage response (DDR)-mechanisms. Remarkably, the homologous recombination (HR) is the major DSBs repairing pathway, in which RAD52 gene has a crucial repairing role by promoting the annealing of complementary single-stranded DNA and by stimulating RAD51 recombinase activity. Evidence suggests that variations in RA...
Suppression of the Double-Strand-Break-Repair Defect of the Saccharomyces cerevisiae rad57 Mutant
Genetics, 2009
The Rad51 paralogs Rad55 and Rad57 form a heterodimer required to mediate the formation and/or stabilization of the Rad51 filament. To further characterize the function of Rad55-Rad57, we used a combination of rad57 partial suppressors to determine whether the DNA repair and recombination defects of the rad57 mutant could be completely suppressed. The combination of all suppressors, elevated temperature, srs2, rad51-I345T, and mating-type (MAT) heterozygosity resulted in almost complete suppression of the rad57 mutant defect in the recruitment of Rad51 to DNA-damaged sites, as well as survival in response to ionizing radiation and camptothecin. In a physical assay to monitor the kinetics of double-strand-break (DSB)-induced gene conversion, the rad57 mutant defect was effectively suppressed by srs2 and MAT heterozygosity, but these same suppressors failed to suppress the spontaneous recombination defect. Thus the Rad55-Rad57 heterodimer appears to have a unique function in spontaneo...
Molecular and Cellular Biology, 2006
Homologous recombination is a versatile DNA damage repair pathway requiring Rad51 and Rad54. Here we show that a mammalian Rad54 paralog, Rad54B, displays physical and functional interactions with Rad51 and DNA that are similar to those of Rad54. While ablation of Rad54 in mouse embryonic stem (ES) cells leads to a mild reduction in homologous recombination efficiency, the absence of Rad54B has little effect. However, the absence of both Rad54 and Rad54B dramatically reduces homologous recombination efficiency. Furthermore, we show that Rad54B protects ES cells from ionizing radiation and the interstrand DNA cross-linking agent mitomycin C. Interestingly, at the ES cell level the paralogs do not display an additive or synergic interaction with respect to mitomycin C sensitivity, yet animals lacking both Rad54 and Rad54B are dramatically sensitized to mitomycin C compared to either single mutant. This suggests that the paralogs possibly function in a tissue-specific manner. Finally, we show that Rad54, but not Rad54B, is needed for a normal distribution of Rad51 on meiotic chromosomes. Thus, even though the paralogs have similar biochemical properties, genetic analysis in mice uncovered their nonoverlapping roles.