scholarly journals The essential helicase gene RAD3 suppresses short-sequence recombination in Saccharomyces cerevisiae.

1995 ◽  
Vol 15 (8) ◽  
pp. 3998-4008 ◽  
Author(s):  
A M Bailis ◽  
S Maines ◽  
M T Negritto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Physical Stability ◽  
Strand Break ◽  
Gene Replacement ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Homologous Sequences ◽  
Helicase Gene ◽  
Mutant Cells

We have isolated an allele of the essential DNA repair and transcription gene RAD3 that relaxes the restriction against recombination between short DNA sequences in Saccharomyces cerevisiae. Double-strand break repair and gene replacement events requiring recombination between short identical or mismatched sequences were stimulated in the rad3-G595R mutant cells. We also observed an increase in the physical stability of double-strand breaks in the rad3-G595R mutant cells. These results suggest that the RAD3 gene suppresses recombination involving short homologous sequences by promoting the degradation of the ends of broken DNA molecules.

scholarly journals Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events

1994 ◽  
Vol 14 (2) ◽  
pp. 1293-1301
Author(s):  
K M Kramer ◽  
J A Brock ◽  
K Bloom ◽  
J K Moore ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cleavage Site ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
High Level ◽  
A Site ◽  
Mat Locus ◽  
Mechanical Rupture

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 MAT 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 (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells.

scholarly journals Mutations in the Saccharomyces cerevisiae CDC1 gene affect double-strand-break-induced intrachromosomal recombination.

1994 ◽  
Vol 14 (12) ◽  
pp. 8037-8050 ◽  
Author(s):  
J Halbrook ◽  
M F Hoekstra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Intrachromosomal Recombination ◽  
Spontaneous Frequency ◽  
Type Strains

To isolate Saccharomyces cerevisiae mutants defective in recombinational DNA repair, we constructed a strain that contains duplicated ura3 alleles that flank LEU2 and ADE5 genes at the ura3 locus on chromosome V. When a HO endonuclease cleavage site is located within one of the ura3 alleles, Ura+ recombination is increased over 100-fold in wild-type strains following HO induction from the GAL1, 10 promoter. This strain was used to screen for mutants that exhibited reduced levels of HO-induced intrachromosomal recombination without significantly affecting the spontaneous frequency of Ura+ recombination. One of the mutations isolated through this screen was found to affect the essential gene CDC1. This mutation, cdc1-100, completely eliminated HO-induced Ura+ recombination yet maintained both spontaneous preinduced recombination levels and cell viability, cdc1-100 mutants were moderately sensitive to killing by methyl methanesulfonate and gamma irradiation. The effect of the cdc1-100 mutation on recombinational double-strand break repair indicates that a recombinationally silent mechanism other than sister chromatid exchange was responsible for the efficient repair of DNA double-strand breaks.

scholarly journals Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

1994 ◽  
Vol 14 (2) ◽  
pp. 1293-1301 ◽  
Author(s):  
K M Kramer ◽  
J A Brock ◽  
K Bloom ◽  
J K Moore ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cleavage Site ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
High Level ◽  
A Site ◽  
Mat Locus ◽  
Mechanical Rupture

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 MAT 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 (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells.

scholarly journals Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae.

1995 ◽  
Vol 15 (4) ◽  
pp. 1968-1973 ◽  
Author(s):  
A Choulika ◽  
A Perrin ◽  
B Dujon ◽  
J F Nicolas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Mouse Genome ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Chromosomal Dna ◽  
Recognition Sequence ◽  
Strand Breaks ◽  
Low Probability ◽  
Active Promoter

The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous regions flanking the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter. As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break. This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells. These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements.

scholarly journals Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (5) ◽  
pp. 3414-3425 ◽  
Author(s):  
E L Ivanov ◽  
N Sugawara ◽  
C I White ◽  
F Fabre ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Strand Break ◽  
Strand Breaks ◽  
Acid Protein ◽  
Mating Type Switching ◽  
One Step ◽  
Mitotic Gene Conversion ◽  
Gene Switching ◽  
Mutant Cells

In Saccharomyces cerevisiae, a large number of genes in the RAD52 epistasis group has been implicated in the repair of chromosomal double-strand breaks and in both mitotic and meiotic homologous recombination. While most of these genes are essential for yeast mating-type (MAT) gene switching, neither RAD50 nor XRS2 is required to complete this specialized mitotic gene conversion process. Using a galactose-inducible HO endonuclease gene to initiate MAT switching, we have examined the effect of null mutations of RAD50 and of XRS2 on intermediate steps of this recombination event. Both rad50 and xrs2 mutants exhibit a marked delay in the completion of switching. Both mutations reduce the extent of 5'-to-3' degradation from the end of the HO-created double-strand break. The steps of initial strand invasion and new DNA synthesis are delayed by approximately 30 min in mutant cells. However, later events are still further delayed, suggesting that XRS2 and RAD50 affect more than one step in the process. In the rad50 xrs2 double mutant, the completion of MAT switching is delayed more than in either single mutant, without reducing the overall efficiency of the process. The XRS2 gene encodes an 854-amino-acid protein with no obvious similarity to the Rad50 protein or to any other protein in the database. Overexpression of RAD50 does not complement the defects in xrs2 or vice versa.

scholarly journals Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11

2005 ◽  
Vol 171 (5) ◽  
pp. 765-771 ◽  
Author(s):  
Michela Di Virgilio ◽  
Jean Gautier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Mrn Complex ◽  
Nhej Repair ◽  
Kinetics Of

Mre11–Rad50–Nbs1 (MRN) complex involvement in nonhomologous end joining (NHEJ) is controversial. The MRN complex is required for NHEJ in Saccharomyces cerevisiae but not in Schizosaccharomyces pombe. In vertebrates, Mre11, Rad50, and Nbs1 are essential genes, and studies have been limited to cells carrying hypomorphic mutations in Mre11 or Nbs1, which still perform several MRN complex–associated activities. In this study, we analyze the effects of Mre11 loss on the mechanism of vertebrate NHEJ by using a chromatinized plasmid double-strand break (DSB) repair assay in cell-free extracts from Xenopus laevis. Mre11-depleted extracts are able to support efficient NHEJ repair of DSBs regardless of the end structure. Mre11 depletion does not alter the kinetics of end joining or the type and frequency of junctions found in repaired products. Finally, Ku70-independent end-joining events are not affected by Mre11 loss. Our data demonstrate that the MRN complex is not required for efficient and accurate NHEJ-mediated repair of DSBs in this vertebrate system.

scholarly journals Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (5) ◽  
pp. 2164-2173 ◽  
Author(s):  
J K Moore ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Strand Breaks ◽  
Large Deletions ◽  
Nhej Repair

In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end joining (NHEJ) that closely resemble events in mammalian cells. In one pathway the chromosome ends are degraded to yield deletions with different sizes whose endpoints have 1 to 6 bp of homology. Alternatively, the 4-bp overhanging 3' ends of HO-cut DNA (5'-AACA-3') are not degraded but can be base paired in misalignment to produce +CA and +ACA insertions. When HO was expressed throughout the cell cycle, the efficiency of NHEJ repair was 30 times higher than when HO was expressed only in G1. The types of repair events were also very different when HO was expressed throughout the cell cycle; 78% of survivors had small insertions, while almost none had large deletions. When HO expression was confined to the G1 phase, only 21% were insertions and 38% had large deletions. These results suggest that there are distinct mechanisms of NHEJ repair producing either insertions or deletions and that these two pathways are differently affected by the time in the cell cycle when HO is expressed. The frequency of NHEJ is unaltered in strains from which RAD1, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, deletions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold when HO was not cell cycle regulated. Moreover, mutations in these three genes markedly reduced +CA insertions, while significantly increasing the proportion of both small (-ACA) and larger deletion events. In contrast, the rad5O mutation had little effect on the viability of G1-induced cells but significantly reduced the frequency of both +CA insertions and -ACA deletions in favor of larger deletions. Thus, RAD50 (and by extension XRS2 and MRE11) exerts a much more important role in the insertion-producing pathway of NHEJ repair found in S and/or G2 than in the less frequent deletion events that predominate when HO is expressed only in G1.

Mutations in the Saccharomyces cerevisiae CDC1 gene affect double-strand-break-induced intrachromosomal recombination

1994 ◽  
Vol 14 (12) ◽  
pp. 8037-8050
Author(s):  
J Halbrook ◽  
M F Hoekstra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Intrachromosomal Recombination ◽  
Spontaneous Frequency ◽  
Type Strains

To isolate Saccharomyces cerevisiae mutants defective in recombinational DNA repair, we constructed a strain that contains duplicated ura3 alleles that flank LEU2 and ADE5 genes at the ura3 locus on chromosome V. When a HO endonuclease cleavage site is located within one of the ura3 alleles, Ura+ recombination is increased over 100-fold in wild-type strains following HO induction from the GAL1, 10 promoter. This strain was used to screen for mutants that exhibited reduced levels of HO-induced intrachromosomal recombination without significantly affecting the spontaneous frequency of Ura+ recombination. One of the mutations isolated through this screen was found to affect the essential gene CDC1. This mutation, cdc1-100, completely eliminated HO-induced Ura+ recombination yet maintained both spontaneous preinduced recombination levels and cell viability, cdc1-100 mutants were moderately sensitive to killing by methyl methanesulfonate and gamma irradiation. The effect of the cdc1-100 mutation on recombinational double-strand break repair indicates that a recombinationally silent mechanism other than sister chromatid exchange was responsible for the efficient repair of DNA double-strand breaks.

scholarly journals Capture of DNA Sequences at Double-Strand Breaks in Mammalian Chromosomes

Genetics ◽  
2001 ◽  
Vol 158 (4) ◽  
pp. 1665-1674 ◽  
Author(s):  
Yunfu Lin ◽  
Alan S Waldman
Keyword(s):  
Dna Sequences ◽  
Strand Break ◽  
Endogenous Retrovirus ◽  
Repeat Sequence ◽  
Double Strand Breaks ◽  
Induced Mutations ◽  
Strand Breaks ◽  
Dna Capture ◽  
Intracisternal A Particle ◽  
Dna Substrate

Abstract To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. To introduce a genomic DSB, cells were electroporated with a plasmid expressing endonuclease I-SceI, and clones that had lost tk function were selected. Among 253 clones analyzed, 78% displayed small deletions or insertions of several nucleotides at the DSB site. Surprisingly, ~8% of recovered mutations involved the capture of one or more DNA fragments. Among 21 clones that had captured DNA, 10 harbored a specific segment of the I-SceI expression plasmid mapping between two replication origins on the plasmid. Four clones had captured a long terminal repeat sequence from an intracisternal A particle (an endogenous retrovirus-like sequence) and one had captured what appears to be a cDNA copy of a moderately repetitive B2 sequence. Additional clones displayed segments of the tk gene and/or microsatellite sequences copied into the DSB. This first systematic study of DNA capture at DSBs in a mammalian genome suggests that DSB repair may play a considerable role in the evolution of eukaryotic genomes.

scholarly journals Homology Search and Choice of Homologous Partner during Mitotic Recombination

1999 ◽  
Vol 19 (6) ◽  
pp. 4134-4142 ◽  
Author(s):  
Ori Inbar ◽  
Martin Kupiec
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Sequence Divergence ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Repeated Sequences ◽  
Homology Search ◽  
Strand Breaks ◽  
Repair Genes

ABSTRACT Homologous recombination is an important DNA repair mechanism in vegetative cells. During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences (gene conversion). This event is often accompanied by a reciprocal exchange between the homologous molecules, resulting in crossing over. The repair of DNA damage by homologous recombination with repeated sequences dispersed throughout the genome might result in chromosomal aberrations or in the inactivation of genes. It is therefore important to understand how the suitable homologous partner for recombination is chosen. We have developed a system in the yeast Saccharomyces cerevisiae that can monitor the fate of a chromosomal double-strand break without the need to select for recombinants. The broken chromosome is efficiently repaired by recombination with one of two potential partners located elsewhere in the genome. One of the partners has homology to the broken ends of the chromosome, whereas the other is homologous to sequences distant from the break. Surprisingly, a large proportion of the repair is carried out by recombination involving the sequences distant from the broken ends. This repair is very efficient, despite the fact that it requires the processing of a large chromosomal region flanking the break. Our results imply that the homology search involves extensive regions of the broken chromosome and is not carried out exclusively by sequences adjacent to the double-strand break. We show that the mechanism that governs the choice of homologous partners is affected by the length and sequence divergence of the interacting partners, as well as by mutations in the mismatch repair genes. We present a model to explain how the suitable homologous partner is chosen during recombinational repair. The model provides a mechanism that may guard the integrity of the genome by preventing recombination between dispersed repeated sequences.

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