Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks

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.

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A Requirement for Recombinational Repair in Saccharomyces cerevisiae Is Caused by DNA Replication Defects of mec1 Mutants

Genetics ◽

10.1093/genetics/153.2.595 ◽

1999 ◽

Vol 153 (2) ◽

pp. 595-605 ◽

Cited By ~ 2

Author(s):

Bradley J Merrill ◽

Connie Holm

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Synthesis ◽

Synthetic Lethality ◽

Velocity Sedimentation ◽

Recombinational Repair ◽

Repair Pathway ◽

Dna Breaks ◽

Single Stranded Dna ◽

Double Stranded Dna

Abstract To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

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Effects of Temperature on the Meiotic Recombination Landscape of the Yeast Saccharomyces cerevisiae

mBio ◽

10.1128/mbio.02099-17 ◽

2017 ◽

Vol 8 (6) ◽

Author(s):

Ke Zhang ◽

Xue-Chang Wu ◽

Dao-Qiong Zheng ◽

Thomas D. Petes

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Conversion ◽

Meiotic Recombination ◽

Hot Spots ◽

Genetic Maps ◽

Dna Breaks ◽

Low Levels ◽

C 30 ◽

Recombination Hot Spots ◽

In Cells

ABSTRACT Although meiosis in warm-blooded organisms takes place in a narrow temperature range, meiosis in many organisms occurs over a wide variety of temperatures. We analyzed the properties of meiosis in the yeast Saccharomyces cerevisiae in cells sporulated at 14°C, 30°C, or 37°C. Using comparative-genomic-hybridization microarrays, we examined the distribution of Spo11-generated meiosis-specific double-stranded DNA breaks throughout the genome. Although there were between 300 and 400 regions of the genome with high levels of recombination (hot spots) observed at each temperature, only about 20% of these hot spots were found to have occurred independently of the temperature. In S. cerevisiae , regions near the telomeres and centromeres tend to have low levels of meiotic recombination. This tendency was observed in cells sporulated at 14°C and 30°C, but not at 37°C. Thus, the temperature of sporulation in yeast affects some global property of chromosome structure relevant to meiotic recombination. Using single-nucleotide polymorphism (SNP)-specific whole-genome microarrays, we also examined crossovers and their associated gene conversion events as well as gene conversion events that were unassociated with crossovers in all four spores of tetrads obtained by sporulation of diploids at 14°C, 30°C, or 37°C. Although tetrads from cells sporulated at 30°C had slightly (20%) more crossovers than those derived from cells sporulated at the other two temperatures, spore viability was good at all three temperatures. Thus, despite temperature-induced variation in the genetic maps, yeast cells produce viable haploid products at a wide variety of sporulation temperatures. IMPORTANCE In the yeast Saccharomyces cerevisiae , recombination is usually studied in cells that undergo meiosis at 25°C or 30°C. In a genome-wide analysis, we showed that the locations of genomic regions with high and low levels of meiotic recombination (hot spots and cold spots, respectively) differed dramatically in cells sporulated at 14°C, 30°C, and 37°C. Thus, in yeast, and likely in other non-warm-blooded organisms, genetic maps are strongly affected by the environment.

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Numerical and Spatial Patterning of Yeast Meiotic DNA Breaks by Tel1

10.1101/059022 ◽

2016 ◽

Cited By ~ 3

Author(s):

Neeman Mohibullah ◽

Scott Keeney

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Deep Sequencing ◽

Meiotic Recombination ◽

Double Strand Breaks ◽

Genome Integrity ◽

Spatial Patterning ◽

Dna Breaks ◽

Strand Breaks ◽

Context Dependent

AbstractThe Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are dangerous lesions that can disrupt genome integrity, so meiotic cells regulate their number, timing, and distribution. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to examine the contribution of the DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM) to this regulation in Saccharomyces cerevisiae. A tel1Δ mutant had globally increased amounts of Spo11-oligonucleotide complexes and altered Spo11-oligonucleotide lengths, consistent with conserved roles for Tel1 in control of DSB number and processing. A kinase-dead tell mutation also increased Spo11-oligonucleotide levels, but mutating known Tel1 phosphotargets on Hop1 and Rec114 did not. Deep sequencing of Spo11 oligonucleotides from tel1Δ mutants demonstrated that Tel1 shapes the nonrandom DSB distribution in ways that are distinct but partially overlapping with previously described contributions of the recombination regulator Zip3. Finally, we uncover a context-dependent role for Tel1 in hotspot competition, in which an artificial DSB hotspot inhibits nearby hotspots. Evidence for Tel1-dependent competition involving strong natural hotspots is also provided.

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Microhomology Selection for Microhomology Mediated End Joining in Saccharomyces cerevisiae

Genes ◽

10.3390/genes10040284 ◽

2019 ◽

Vol 10 (4) ◽

pp. 284 ◽

Cited By ~ 3

Author(s):

Kihoon Lee ◽

Jae-Hoon Ji ◽

Kihoon Yoon ◽

Jun Che ◽

Ja-Hwan Seol ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Budding Yeast ◽

Product Formation ◽

Dna Breaks ◽

End Joining ◽

Base Pairs ◽

Circular Chromosome ◽

Proximal Side ◽

Selection For ◽

Insight Into

Microhomology-mediated end joining (MMEJ) anneals short, imperfect microhomologies flanking DNA breaks, producing repair products with deletions in a Ku- and RAD52-independent fashion. Puzzlingly, MMEJ preferentially selects certain microhomologies over others, even when multiple microhomologies are available. To define rules and parameters for microhomology selection, we altered the length, the position, and the level of mismatches to the microhomologies flanking homothallic switching (HO) endonuclease-induced breaks and assessed their effect on MMEJ frequency and the types of repair product formation. We found that microhomology of eight to 20 base pairs carrying no more than 20% mismatches efficiently induced MMEJ. Deletion of MSH6 did not impact MMEJ frequency. MMEJ preferentially chose a microhomology pair that was more proximal from the break. Interestingly, MMEJ events preferentially retained the centromere proximal side of the HO break, while the sequences proximal to the telomere were frequently deleted. The asymmetry in the deletional profile among MMEJ products was reduced when HO was induced on the circular chromosome. The results provide insight into how cells search and select microhomologies for MMEJ in budding yeast.

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A comparison and assessment of computational method for identifying recombination hotspots in Saccharomyces cerevisiae

Briefings in Bioinformatics ◽

10.1093/bib/bbz123 ◽

2019 ◽

Vol 21 (5) ◽

pp. 1568-1580 ◽

Cited By ~ 34

Author(s):

Hui Yang ◽

Wuritu Yang ◽

Fu-Ying Dao ◽

Hao Lv ◽

Hui Ding ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Computational Methods ◽

Driving Forces ◽

Biological Evolution ◽

Computational Method ◽

Dna Breaks ◽

Data Set ◽

Recombination Hotspots ◽

Double Strand Dna Breaks ◽

Comprehensive Survey

Abstract Meiotic recombination is one of the most important driving forces of biological evolution, which is initiated by double-strand DNA breaks. Recombination has important roles in genome diversity and evolution. This review firstly provides a comprehensive survey of the 15 computational methods developed for identifying recombination hotspots in Saccharomyces cerevisiae. These computational methods were discussed and compared in terms of underlying algorithms, extracted features, predictive capability and practical utility. Subsequently, a more objective benchmark data set was constructed to develop a new predictor iRSpot-Pse6NC2.0 (http://lin-group.cn/server/iRSpot-Pse6NC2.0). To further demonstrate the generalization ability of these methods, we compared iRSpot-Pse6NC2.0 with existing methods on the chromosome XVI of S. cerevisiae. The results of the independent data set test demonstrated that the new predictor is superior to existing tools in the identification of recombination hotspots. The iRSpot-Pse6NC2.0 will become an important tool for identifying recombination hotspot.

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Saccharomyces cerevisiae pol30(Proliferating Cell Nuclear Antigen) Mutations Impair Replication Fidelity and Mismatch Repair

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7801 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7801-7815 ◽

Cited By ~ 75

Author(s):

Clark Chen ◽

Bradley J. Merrill ◽

Patrick J. Lau ◽

Connie Holm ◽

Richard D. Kolodner

Keyword(s):

Saccharomyces Cerevisiae ◽

Mismatch Repair ◽

Synthetic Lethality ◽

Mutation Rates ◽

Nuclear Antigen ◽

Dependent Manner ◽

Dna Breaks ◽

Group A ◽

Group B

ABSTRACT To understand the role of POL30 in mutation suppression, 11 Saccharomyces cerevisiae pol30 mutator mutants were characterized. These mutants were grouped based on their mutagenic defects. Many pol30 mutants harbor multiple mutagenic defects and were placed in more than one group. Group A mutations (pol30-52, -104, -108, and -126) caused defects in mismatch repair (MMR). These mutants exhibited mutation rates and spectra reminiscent of MMR-defective mutants and were defective in an in vivo MMR assay. The mutation rates of group A mutants were enhanced by a msh2or a msh6 mutation, indicating that MMR deficiency is not the only mutagenic defect present. Group B mutants (pol30-45, -103, -105, -126, and -114) exhibited increased accumulation of either deletions alone or a combination of deletions and duplications (4 to 60 bp). All deletion and duplication breakpoints were flanked by 3 to 7 bp of imperfect direct repeats. Genetic analysis of one representative group B mutant, pol30-126, suggested polymerase slippage as the likely mutagenic mechanism. Group C mutants (pol30-100, -103, -105, -108, and -114) accumulated base substitutions and exhibited synergistic increases in mutation rate when combined withmsh6 mutations, suggesting increased DNA polymerase misincorporation as a mutagenic defect. The synthetic lethality between a group A mutant, pol30-104, and rad52 was almost completely suppressed by the inactivation of MSH2. Moreover, pol30-104 caused a hyperrecombination phenotype that was partially suppressed by a msh2 mutation. These results suggest that pol30-104 strains accumulate DNA breaks in a MSH2-dependent manner.

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Loss of a histone deacetylase dramatically alters the genomic distribution of Spo11p-catalyzed DNA breaks in Saccharomyces cerevisiae

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0700412104 ◽

2007 ◽

Vol 104 (10) ◽

pp. 3955-3960 ◽

Cited By ~ 54

Author(s):

P. A. Mieczkowski ◽

M. Dominska ◽

M. J. Buck ◽

J. D. Lieb ◽

T. D. Petes

Keyword(s):

Saccharomyces Cerevisiae ◽

Histone Deacetylase ◽

Dna Breaks ◽

Genomic Distribution

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

Genetics ◽

10.1093/genetics/158.4.1457 ◽

2001 ◽

Vol 158 (4) ◽

pp. 1457-1476 ◽

Cited By ~ 5

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.

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Siz2 Prevents Ribosomal DNA Recombination by Modulating Levels of Tof2 in Saccharomyces cerevisiae

mSphere ◽

10.1128/msphere.00713-19 ◽

2019 ◽

Vol 4 (6) ◽

Author(s):

Neethu Maria Abraham ◽

Kathirvel Ramalingam ◽

Saketh Murthy ◽

Krishnaveni Mishra

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Dna ◽

Posttranslational Modifications ◽

Dna Recombination ◽

Dna Breaks ◽

Direct Role ◽

Content Type ◽

Repetitive Nature ◽

The Stability ◽

Consequent Increase

ABSTRACT Ribosomal DNA (rDNA) recombination in budding yeast is regulated by multiple converging processes, including posttranslational modifications such as SUMOylation. In this study, we report that the absence of a SUMO E3 ligase, Siz2, results in increased unequal rDNA exchange. We show that Siz2 is enriched at the replication fork barrier (RFB) in the rDNA and also controls the homeostasis of Tof2 protein. siz2Δ resulted in increased accumulation of total Tof2 in the cell and a consequent increase in the enrichment of Tof2 at the rDNA. Overproducing Tof2 ectopically or conditional overexpression of Tof2 also resulted in higher levels of rDNA recombination, suggesting a direct role for Tof2. Additionally, our chromatin immunoprecipitation (ChIP) data indicate that the accumulation of Tof2 in a siz2Δ mutant resulted in an enhanced association of Fob1, an RFB binding protein at the rDNA at the RFB. This increased Fob1 association at the RFB may have resulted in the elevated rDNA recombination. Our study thus demonstrates that the Tof2 levels modulate recombination at the rDNA. IMPORTANCE The genes that encode rRNA in Saccharomyces cerevisiae are organized as multiple repeats. The repetitive nature and heavy transcription of this region make it prone to DNA breaks. DNA breaks could lead to recombination, which could result in either loss or gain of repeats with detrimental consequences to the cell. Multiple mechanisms operate to maintain the stability of rDNA. Earlier studies reported that the absence of Ulp2, a deSUMOylase, resulted in declining levels of Tof2 and thereby disrupted rDNA silencing. In contrast, our findings suggest that accumulation of Tof2 can also result in increased rDNA recombination, through a mechanism that involves Fob1, an RFB-bound protein. While our study has examined only Tof2, rDNA recombination could be regulated by other proteins through a mechanism similar to this.

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