scholarly journals Meiotic Recombination Involving Heterozygous Large Insertions inSaccharomyces cerevisiae: Formation and Repair of Large, Unpaired DNA Loops

Genetics ◽  
2001 ◽  
Vol 158 (4) ◽  
pp. 1457-1476 ◽  
Author(s):  
Hutton M Kearney ◽  
David T Kirkpatrick ◽  
Jennifer L Gerton ◽  
Thomas D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Excision Repair ◽  
Large Bubble ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Dna Loop ◽  
Nucleotide Excision ◽  
Dna Strands ◽  
Mmr Proteins

AbstractMeiotic recombination in Saccharomyces cerevisiae involves the formation of heteroduplexes, duplexes containing DNA strands derived from two different homologues. If the two strands of DNA differ by an insertion or deletion, the heteroduplex will contain an unpaired DNA loop. We found that unpaired loops as large as 5.6 kb can be accommodated within a heteroduplex. Repair of these loops involved the nucleotide excision repair (NER) enzymes Rad1p and Rad10p and the mismatch repair (MMR) proteins Msh2p and Msh3p, but not several other NER (Rad2p and Rad14p) and MMR (Msh4p, Msh6p, Mlh1p, Pms1p, Mlh2p, Mlh3p) proteins. Heteroduplexes were also formed with DNA strands derived from alleles containing two different large insertions, creating a large “bubble”; repair of this substrate was dependent on Rad1p. Although meiotic recombination events in yeast are initiated by double-strand DNA breaks (DSBs), we showed that DSBs occurring within heterozygous insertions do not stimulate interhomologue recombination.

scholarly journals Competition Between Adjacent Meiotic Recombination Hotspots in the Yeast Saccharomyces cerevisiae

Genetics ◽  
1997 ◽  
Vol 145 (3) ◽  
pp. 661-670 ◽  
Author(s):  
Qing-Qing Fan ◽  
Fei Xu ◽  
Michael A White ◽  
Thomas D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Telomeric Sequence ◽  
Coding Region ◽  
Dna Breaks ◽  
Local Formation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Recombination Hotspots ◽  
Double Strand Dna Breaks ◽  
His4 Gene

In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes.

scholarly journals Global Analysis of the Relationship between the Binding of the Bas1p Transcription Factor and Meiosis-Specific Double-Strand DNA Breaks in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (3) ◽  
pp. 1014-1027 ◽  
Author(s):  
Piotr A. Mieczkowski ◽  
Margaret Dominska ◽  
Michael J. Buck ◽  
Jennifer L. Gerton ◽  
Jason D. Lieb ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Meiotic Recombination ◽  
Binding Sites ◽  
Hot Spot ◽  
Wild Type ◽  
Dna Breaks ◽  
Recombination Activity ◽  
Double Strand Dna Breaks ◽  
Spot Activity

ABSTRACT In the yeast Saccharomyces cerevisiae, certain genomic regions have very high levels of meiotic recombination (hot spots). The hot spot activity associated with the HIS4 gene requires the Bas1p transcription factor. To determine whether this relationship between transcription factor binding and hot spot activity is general, we used DNA microarrays to map all genomic Bas1p binding sites and to map the frequency of meiosis-specific double-strand DNA breaks (as an estimate of the recombination activity) of all genes in both wild-type and bas1 strains. We identified sites of Bas1p-DNA interactions upstream of 71 genes, many of which are involved in histidine and purine biosynthesis. Our analysis of recombination activity in wild-type and bas1 strains showed that the recombination activities of some genes with Bas1p binding sites were dependent on Bas1p (as observed for HIS4), whereas the activities of other genes with Bas1p binding sites were unaffected or were repressed by Bas1p. These data demonstrate that the effect of transcription factors on meiotic recombination activity is strongly context dependent. In wild-type and bas1 strains, meiotic recombination was strongly suppressed in large (25- to 150-kb) chromosomal regions near the telomeres and centromeres and in the region flanking the rRNA genes. These results argue that both local and regional factors affect the level of meiotic recombination.

scholarly journals Double mutants of Saccharomyces cerevisiae with alterations in global genome and transcription-coupled repair.

1996 ◽  
Vol 16 (2) ◽  
pp. 496-502 ◽  
Author(s):  
R A Verhage ◽  
A J van Gool ◽  
N de Groot ◽  
J H Hoeijmakers ◽  
P van de Putte ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Excision Repair ◽  
Template Strand ◽  
Nucleotide Excision ◽  
Dna Strands ◽  
Repair Activity ◽  
Partial Overlap ◽  
Global Genome Repair ◽  
Transcription Coupled Repair ◽  
Genome Repair

The nucleotide excision repair (NER) pathway is thought to consist of two subpathways: transcription-coupled repair, limited to the transcribed strand of active genes, and global genome repair for nontranscribed DNA strands. Recently we cloned the RAD26 gene, the Saccharomyces cerevisiae homolog of human CSB/ERCC6, a gene involved in transcription-coupled repair and the disorder Cockayne syndrome. This paper describes the analysis of yeast double mutants selectively affected in each NER subpathway. Although rad26 disruption mutants are defective in transcription-coupled repair, they are not UV sensitive. However, double mutants of RAD26 with the global genome repair determinants RAD7 and RAD16 appeared more UV sensitive than the single rad7 or rad16 mutants but not as sensitive as completely NER-deficient mutants. These findings unmask a role of RAD26 and transcription-coupled repair in UV survival, indicate that transcription-coupled repair and global genome repair are partially overlapping, and provide evidence for a residual NER modality in the double mutants. Analysis of dimer removal from the active RPB2 gene in the rad7/16 rad26 double mutants revealed (i) a contribution of the global genome repair factors Rad7p and Rad16p to repair of the transcribed strand, confirming the partial overlap between both NER subpathways, and (ii) residual repair specifically of the transcribed strand. To investigate the transcription dependence of this repair activity, strand-specific repair of the inducible GAL7 gene was investigated. The template strand of this gene was repaired only under induced conditions, pointing to a role for transcription in the residual repair in the double mutants and suggesting that transcription-coupled repair can to some extent operate independently from Rad26p. Our findings also indicate locus heterogeneity for the dependence of transcription-coupled repair on RAD26.

scholarly journals Factors that affect the location and frequency of meiosis-induced double-strand breaks in Saccharomyces cerevisiae.

Genetics ◽  
1995 ◽  
Vol 140 (1) ◽  
pp. 55-66 ◽  
Author(s):  
T C Wu ◽  
M Lichten
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Structure ◽  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Yeast Genome ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Double Strand Dna Breaks ◽  
A Site ◽  
Chromosomal Sequences

Abstract Double-strand DNA breaks (DSBs) initiate meiotic recombination in Saccharomyces cerevisiae. DSBs occur at sites that are hypersensitive in nuclease digests of chromatin, suggesting a role for chromatin structure in determining DSB location. We show here that the frequency of DSBs at a site is not determined simply by DNA sequence or by features of chromatin structure. An arg4-containing plasmid was inserted at several different locations in the yeast genome. Meiosis-induced DSBs occurred at similar sites in pBR322-derived portions of the construct at all insert loci, and the frequency of these breaks varied in a manner that mirrored the frequency of meiotic recombination in the arg4 portion of the insert. However, DSBs did not occur in the insert-borne arg4 gene at a site that is frequently broken at the normal ARG4 locus, even though the insert-borne arg4 gene and the normal ARG4 locus displayed similar DNase I hypersensitivity patterns. Deletions that removed active DSB sites from an insert at HIS4 restored breaks to the insert-borne arg4 gene and to a DSB site in flanking chromosomal sequences. We conclude that the frequency of DSB at a site can be affected by sequences several thousand nucleotides away and suggest that this is because of competition between DSB sites for locally limited factors.

A Mutation in a Saccharomyces cerevisiae Gene (RAD3) Required for Nucleotide Excision Repair and Transcription Increases the Efficiency of Mismatch Correction

Genetics ◽  
1996 ◽  
Vol 144 (2) ◽  
pp. 459-466 ◽  
Author(s):  
Yingying Yang ◽  
Anthony L Johnson ◽  
Leland H Johnston ◽  
Wolfram Siede ◽  
Errol C Friedberg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Excision Repair ◽  
Spontaneous Mutation ◽  
Surprising Result ◽  
Spontaneous Mutation Rate ◽  
Mismatch Correction ◽  
Nucleotide Excision ◽  
Repair Genes ◽  
Type Strains

Abstract RAD3 functions in DNA repair and transcription in Saccharomyces cerevisiae and particular rad3 alleles confer a mutator phenotype, possibly as a consequence of defective mismatch correction. We assessed the potential involvement of the Rad3 protein in mismatch correction by comparing heteroduplex repair in isogenic rad3-1 and wild-type strains. The rad3-1 allele increased the spontaneous mutation rate but did not prevent heteroduplex repair or bias its directionality. Instead, the efficiency of mismatch correction was enhanced in the rad3-1 strain. This surprising result prompted us to examine expression of yeast mismatch repair genes. We determined that MSH2, but not MLH1, is transcriptionally regulated during the cell-cycle like PMSl, and that rad3-1 does not increase the transcript levels for these genes in log phase cells. These observations suggest that the rad3-1 mutation gives rise to an enhanced efficiency of mismatch correction via a process that does not involve transcriptional regulation of mismatch repair. Interestingly, mismatch repair also was more efficient when error-editing by yeast DNA polymerase δ was eliminated. We discuss our results in relation to possible mechanisms that may link the rad3-1 mutation to mismatch correction efficiency.

scholarly journals Fine-Structure Mapping of Meiosis-Specific Double-Strand DNA Breaks at a Recombination Hotspot Associated With an Insertion of Telomeric Sequences Upstream of the HIS4 Locus in Yeast

Genetics ◽  
1996 ◽  
Vol 143 (3) ◽  
pp. 1115-1125 ◽  
Author(s):  
Fei Xu ◽  
Thomas D Petes
Keyword(s):  
Meiotic Recombination ◽  
Hydroxyl Groups ◽  
Sequence Specificity ◽  
Structure Mapping ◽  
Dna Breaks ◽  
Exonuclease Iii ◽  
Recombination Hotspot ◽  
Telomeric Sequences ◽  
Double Strand Dna Breaks ◽  
Double Strand Dna

Abstract Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand DNA breaks (DSBs). Using two approaches, we mapped the position of DSBs associated with a recombination hotspot created by insertion of telomeric sequences into the region upstream of HIS4. We found that the breaks have no obvious sequence specificity and localize to a region of ~50 bp adjacent to the telomeric insertion. By mapping the breaks and by studies of the exonuclease III sensitivity of the broken ends, we conclude that most of the broken DNA molecules have blunt ends with 3′-hydroxyl groups.

scholarly journals Regulation of Mitotic Homeologous Recombination in Yeast: Functions of Mismatch Repair and Nucleotide Excision Repair Genes

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 133-146 ◽  
Author(s):  
Ainsley Nicholson ◽  
Miyono Hendrix ◽  
Sue Jinks-Robertson ◽  
Gray F Crouse
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Mitotic Recombination ◽  
Inverted Repeats ◽  
Replication Errors ◽  
Nucleotide Excision ◽  
Homeologous Recombination ◽  
Repair Genes

Abstract The Saccharomyces cerevisiae homologs of the bacterial mismatch repair proteins MutS and MutL correct replication errors and prevent recombination between homeologous (nonidentical) sequences. Previously, we demonstrated that Msh2p, Msh3p, and Pms1p regulate recombination between 91% identical inverted repeats, and here use the same substrates to show that Mlh1p and Msh6p have important antirecombination roles. In addition, substrates containing defined types of mismatches (base-base mismatches; 1-, 4-, or 12-nt insertion/deletion loops; or 18-nt palindromes) were used to examine recognition of these mismatches in mitotic recombination intermediates. Msh2p was required for recognition of all types of mismatches, whereas Msh6p recognized only base-base mismatches and 1-nt insertion/deletion loops. Msh3p was involved in recognition of the palindrome and all loops, but also had an unexpected antirecombination role when the potential heteroduplex contained only base-base mismatches. In contrast to their similar antimutator roles, Pms1p consistently inhibited recombination to a lesser degree than did Msh2p. In addition to the yeast MutS and MutL homologs, the exonuclease Exo1p and the nucleotide excision repair proteins Rad1p and Rad10p were found to have roles in inhibiting recombination between mismatched substrates.

scholarly journals Photolyases from Saccharomyces cerevisiae and Escherichia coli recognize common binding determinants in DNA containing pyrimidine dimers.

1989 ◽  
Vol 9 (11) ◽  
pp. 4777-4788 ◽  
Author(s):  
M Baer ◽  
G B Sancar
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Spectroscopic Studies ◽  
Pyrimidine Dimer ◽  
Nucleotide Sequence Analysis ◽  
Binding Motif ◽  
Pyrimidine Dimers ◽  
Nucleotide Excision

DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. The results of nucleotide sequence analysis and spectroscopic studies demonstrated that photolyases from Saccharomyces cerevisiae and Escherichia coli share 37% amino acid sequence homology and contain identical chromophores. Do the similarities between these two enzymes extend to their interactions with DNA containing pyrimidine dimers, or does the organization of DNA into nucleosomes in S. cerevisiae necessitate alternative or additional recognition determinants? To answer this question, we used chemical and enzymatic techniques to identify the contacts made on DNA by S. cerevisiae photolyase when it is bound to a pyrimidine dimer and compared these contacts with those made by E. coli photolyase and by a truncated derivative of the yeast enzyme when bound to the same substrate. We found evidence for a common set of interactions between the photolyases and specific phosphates in the backbones of both strands as well as for interactions with bases in both the major and minor grooves of dimer-containing DNA. Superimposed on this common pattern were significant differences in the contributions of specific contacts to the overall binding energy, in the interactions of the enzymes with groups on the complementary strand, and in the extent to which other DNA-binding proteins were excluded from the region around the dimer. These results provide strong evidence both for a conserved dimer-binding motif and for the evolution of new interactions that permit photolyases to also act as accessory proteins in nucleotide excision repair. The locations of the specific contacts made by the yeast enzyme indicate that the mechanism of nucleotide excision repair in this organism involves incision(s) at a distance from the pyrimidine dimer.

scholarly journals A Synthetic Interaction between CDC20 and RAD4 in Saccharomyces cerevisiae upon UV Irradiation

2014 ◽  
Vol 2014 ◽  
pp. 1-8
Author(s):  
Bernadette Connors ◽  
Lauren Rochelle ◽  
Asela Roberts ◽  
Graham Howard
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Excision Repair ◽  
Uv Irradiation ◽  
Double Mutant ◽  
Excision Repair ◽  
Strand Break ◽  
Anaphase Promoting Complex ◽  
End Joining ◽  
Ubiquitin Ligase Complex ◽  
Nucleotide Excision

Regulation of DNA repair can be achieved through ubiquitin-mediated degradation of transiently induced proteins. In Saccharomyces cerevisiae, Rad4 is involved in damage recognition during nucleotide excision repair (NER) and, in conjunction with Rad23, recruits other proteins to the site of damage. We identified a synthetic interaction upon UV exposure between Rad4 and Cdc20, a protein that modulates the activity of the anaphase promoting complex (APC/C), a multisubunit E3 ubiquitin ligase complex. The moderately UV sensitive Δrad4 strain became highly sensitive when cdc20-1 was present, and was rescued by overexpression of CDC20. The double mutant is also deficient in elicting RNR3-lacZ transcription upon exposure to UV irradiation or 4-NQO compared with the Δrad4 single mutant. We demonstrate that the Δrad4/cdc20-1 double mutant is defective in double strand break repair by way of a plasmid end-joining assay, indicating that Rad4 acts to ensure that damaged DNA is repaired via a Cdc20-mediated mechanism. This study is the first to present evidence that Cdc20 may play a role in the degradation of proteins involved in nucleotide excision repair.

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