DNA damage-inducible and RAD52-independent repair of DNA double-strand breaks in Saccharomyces cerevisiae (original) (raw)
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Molecular and General Genetics, 1976
With the use of neutral sucrose sedimentation techniques, the size of unirradiated nuclear DNA and the repair of double-strand breaks induced in it by ionizing radiation have been determined in both wild-type and homozygous rad52 diploids of the yeast Saccharomyces cerevisiae. The number average molecular weight of unirradiated DNA in these experiments is 3.0×108±0.3 Daltons. Double-strand breaks are induced with a frequency of 0.58×10-10 per Daltonkrad in the range of 25 to 100 krad. Since repair at low doses is observed in wild-type but not homozygous rad52 strains, the corresponding rad52 gene product is concluded to have a role in the repair process. Cycloheximide was also observed to inhibit repair to a limited extent indicating a requirement for protein synthesis. Based on the sensitivity of various mutants and the induction frequency of double-strand breaks, it is concluded that there are 1 to 2 double-strand breaks per lethal event in diploid cells incapable of repairing these breaks.
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...
Modulation of Saccharomyces cerevisiae DNA double-strand break repair by SRS2 and RAD51
Genetics, 1995
RAD52 function is required for virtually all DNA double-strand break repair and recombination events in Saccharomyces cerevisiae. To gain greater insight into the mechanism of RAD52-mediated repair, we screened for genes that suppress partially active alleles of RAD52 when mutant or overexpressed. Described here is the isolation of a phenotypic null allele of SRS2 that suppressed multiple alleles of RAD52 (rad52B, rad52D, rad52-1 and KlRAD52) and RAD51 (KlRAD51) but failed to suppress either a rad52 delta or a rad51 delta. These results indicate that SRS2 antagonizes RAD51 and RAD52 function in recombinational repair. The mechanism of suppression of RAD52 alleles by srs2 is distinct from that which has been previously described for RAD51 overexpression, as both conditions were shown to act additively with respect to the rad52B allele. Furthermore, overexpression of either RAD52 or RAD51 enhanced the recombination-dependent sensitivity of an srs2 delta RAD52 strain, suggesting that R...
Aberrant Double-Strand Break Repair in rad51 Mutants of Saccharomyces cerevisiae
Molecular and Cellular Biology, 2000
A number of studies of Saccharomyces cerevisiae have revealed RAD51 -independent recombination events. These include spontaneous and double-strand break-induced recombination between repeated sequences, and capture of a chromosome arm by break-induced replication. Although recombination between inverted repeats is considered to be a conservative intramolecular event, the lack of requirement for RAD51 suggests that repair can also occur by a nonconservative mechanism. We propose a model for RAD51 -independent recombination by one-ended strand invasion coupled to DNA synthesis, followed by single-strand annealing. The Rad1/Rad10 endonuclease is required to trim intermediates formed during single-strand annealing and thus was expected to be required for RAD51 -independent events by this model. Double-strand break repair between plasmid-borne inverted repeats was less efficient in rad1 rad51 double mutants than in rad1 and rad51 strains. In addition, repair events were delayed and frequ...
Journal of Biological Physics, 2008
DNA repair, checkpoint pathways and protection mechanisms against different types of perturbations are critical factors for the prevention of genomic instability. The aim of the present work was to analyze the roles of RAD17 and HDF1 gene products during the late stationary phase, in haploid and diploid yeast cells upon gamma irradiation. The checkpoint protein, Rad17, is a component of a PCNA-like complex-the Rad17/Mec3/Ddc1 clamp-acting as a damage sensor; this protein is also involved in double-strand break (DBS) repair in cycling cells. The HDF1 gene product is a key component of the non-homologous end-joining pathway (NHEJ). Diploid and haploid rad17 /rad17 , and hdf1 Saccharomyces cerevisiae mutant strains and corresponding isogenic wild types were used in the present study. Yeast cells were grown in standard liquid nutrient medium, and maintained at 30 • C for 21 days in the stationary phase, without added nutrients. Cell samples were irradiated with 60 Co γ rays at 5 Gy/s, 50 Gy ≤ Dabs ≤ 200 Gy. Thereafter, cells were incubated in PBS (liquid holding: LH, 0 ≤ t ≤ 24 h). DNA chromosomal analysis (by pulsed-field electrophoresis), and surviving fractions were determined as a function of absorbed doses, either immediately after irradiation or after LH. Our results demonstrated that the proteins Rad17, as well as Hdf1, play essential roles in DBS repair and survival after gamma irradiation in the late stationary phase and upon nutrient stress (LH after irradiation). In haploid cells, the main pathway is NHEJ. In the diploid state, the induction of LH recovery requires the function of Rad17. Results are compatible with the action of a network of DBS repair pathways expressed upon different ploidies, and different magnitudes of DNA damage.
… and cellular biology, 1994
In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the AMT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and ifiling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells.
Modulation of Saccharomyces certwkiae DNA Double-Strand Break Repair by SRS2 and RAD51
1995
RAD52 function is required for virtually all DNA double-strand break repair and recombination events in Saccharomyces cereuisiae. To gain greater insight into the mechanism of RAD52-mediated repair, we screened for genes that suppress partially active alleles of RAD52 when mutant or overexpressed. De- scribed here is the isolation of a phenotypic null allele of SRS2 that suppressed multiple alleles of
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
Nucleic Acids Research, 1993
The gene rad22 of the fission yeast Schizosaccharomycespombe has a function in DNA repair and matingtype switching. We have cloned the rad22 gene from a genomic gene bank by functional complementation of the switching defect. An open reading frame coding for a putative protein of 469 amino acids was found by sequence analyses. The rad22 gene contains no intron. A region of 126 amino acids in the N-terminal half of the Rad22 protein has significant homologies (56% identity and 36% similarity) to the Rad52 protein of Saccharomyces cerevisiae. A rad22 disruption strain was constructed which seems to be inviable in a homothallic background. Southern blot analyses have shown that the rad22-67 mutant frequently gives rise to deletions in the mating-type region. These data indicate that the Rad22 protein has a function in the repair of DNA double-strand breaks.