Nuclear oscillations and nuclear filament formation accompany single-strand annealing repair of a dicentric chromosome in Saccharomyces cerevisiae (original) (raw)
Related papers
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
… 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.
Genetics, 1996
The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3 delta mutation has an effect on recombination similar to that of the rad1 delta and rad10 delta mutations. The msh2 delta mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed....
Molecular and cellular biology, 1995
HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1 delta and rad10 delta mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove U...
Healing of Broken Linear Dicentric Chromosomes in Yeast
Genetics, 1984
In yeast, meiotic recombination between a linear chromosome III and a haploid-viable circular chromosome will yield a dicentric, tandemly duplicated chromosome. Spores containing apparently intact dicentric chromosomes were recovered from tetrads with three viable spores. The spore containing the dicentric inherited URA3 (part of the recombinant DNA used to join regions near the ends of the chromosome into a circle) as well as HML, HMR and MAL2 (located near the two ends of a linear but deleted from the circle). The Ura+ Mal+ colonies were highly variegated, giving rise to as many as seven distinctly different stable ("healed") derivatives, some of which were Ura+ Mal+, others Ura+ Mal-and others Ura-Mal' . The colonies were also sectored for five markers (HIS4, LEUZ, CRYI, MAT and THR4) initially heterozygous in the tandemly duplicated dicentric chromosome.-Southern blot and genetic analyses have demonstrated that these stable derivatives arose from mitotic breakage of the dicentric chromosome, followed by one of several different healing events. The majority of the stable derivatives contained circular or linear chromosomes apparently resulting from homologous recombination between a broken chromosome end and a homologous region on the other end of the original dicentric duplicated chromosome. A smaller proportion of events resulted in apparently uniquely healed linear chromosomes in which the broken chromosome acquired a new telomere. In two instances we recovered chromosome 1 1 1 partially duplicated with a novel right end. We have also found one derivative that had also experienced rearrangement of repeated DNA sequences found adjacent to yeast telomeres. ROKEN chromosomes, lacking a telomere, are mitotically unstable in both B maize (MCCLINTOCK 194 1) and the yeast Saccharomyces cerevisiae (MC-CUSKER and HABER 198 1 ; WEIFFENBACH and HABER 198 1 ; HABER, . In both organisms, broken chromosomes can become "healed" (that is, mitotically stable). In maize, such healing events often involved chromosomal rearrangements that in essence allowed a broken chromosome to acquire a new telomere by translocation involving another, nonhomologous chromosome. In other cases, MCCLINTOCK (1 94 1) observed terminally deficient chromosomes in which the origin of the telomeric region could not be determined.
Genetics, 2006
We have previously shown that recombination between 400-bp substrates containing only 4-bp differences, when present in an inverted repeat orientation, is suppressed by >20-fold in wild-type strains of S. cerevisiae. Among the genes involved in this suppression were three genes involved in mismatch repair—MSH2, MSH3, and MSH6—and one in nucleotide excision repair, RAD1. We now report the involvement of these genes in interchromosomal recombination occurring via crossovers using these same short substrates. In these experiments, recombination was stimulated by a double-strand break generated by the HO endonuclease and can occur between completely identical (homologous) substrates or between nonidentical (homeologous) substrates. In addition, a unique feature of this system is that recombining DNA strands can be given a choice of either type of substrate. We find that interchromosomal crossover recombination with these short substrates is severely inhibited in the absence of MSH2, ...
Equal sister chromatid exchange is a major mechanism of double-strand break repair in yeast
Molecular cell, 2003
two sister chromatids. Consequently, either the homologous chromosome or the sister chromatid can serve as the template to repair breaks that occur during S or G2. Evidence of sister chromatid exchange (SCE) has been obtained cytologically in mammalian cells (Sonoda et Universidad de Sevilla 41012 Sevilla al., 1999; Dronkert et al., 2000) and by pulse-field gel electrophoresis of a circular chromosome in yeast meio-Spain sis (Game et al., 1989). Since sister chromatids contain identical DNA sequences, SCE cannot be detected genetically. Some genetic assays based on intrachromo-Summary somal repeat heteroalleles have been developed to detect unequal SCE events (Szostak and Wu, 1980; Equal sister chromatid exchange (SCE) has been thought to be an important mechanism of double-strand Jackson and Fink, 1981; Petes, 1980; Fasullo and Davis, 1988; Fasullo et al., 2001; Klein, 1988; Kadyk and Hart-break (DSB) repair in eukaryotes, but this has never been proven due to the difficulty of distinguishing SCE well, 1992; Johnson and Jasin, 2000), but until now no quantitative method existed to analyze equal SCE. products from parental molecules. To evaluate the biological relevance of equal SCE in DSB repair and to
Recombination Proteins in Yeast
Annual Review of Genetics, 2004
▪ The process of homologous recombination promotes error-free repair of double-strand breaks and is essential for meiosis. 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. 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. Herein, we review recent genetic, biochemical, and structural analyses of the genes and proteins involved in recombination.
Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae
Microbiology and molecular biology reviews : MMBR, 1999
The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination.