scholarly journals Topoisomerases modulate the timing of meiotic DNA breakage and chromosome morphogenesis in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Jonna Heldrich ◽  
Xiaoji Sun ◽  
Luis A. Vale-Silva ◽  
Tovah E. Markowitz ◽  
Andreas Hochwagen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Meiotic Prophase ◽  
Meiotic Chromosome ◽  
Strand Break ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
Meiotic Chromosomes ◽  
Chromosome Synapsis ◽  
Intergenic Regions

AbstractDuring meiotic prophase, concurrent transcription, recombination, and chromosome synapsis, place substantial topological strain on chromosomal DNA, but the role of topoisomerases in this context remains poorly defined. Here, we analyzed the roles topoisomerases I and II (Top1 and Top2) during meiotic prophase in Saccharomyces cerevisiae. We show that both topoisomerases accumulate primarily in promoter-containing intergenic regions of actively transcribing genes. Enrichment partially overlaps meiotic double-strand break (DSB) hotspots, but disruption of either topoisomerase has different effects during meiotic recombination. TOP1 disruption delays DSB induction and shortens the window of DSB accumulation by an unknown mechanism. By contrast, temperature-sensitive top2-1 mutants accumulate DSBs on synapsed chromosomes and exhibit a marked delay in meiotic chromosome remodeling. This defect results from a delay in recruiting the meiotic chromosome remodeler Pch2/TRIP13 but, unexpectedly, is not due to a loss of Top2 catalytic activity. Instead, mutant Top2-1 protein has reduced contact with chromatin but remains associated with meiotic chromosomes, and we provide evidence that this altered binding is responsible for the delay in chromosome remodeling. Our results imply independent roles for topoisomerases I and II in modulating meiotic recombination.

scholarly journals Topoisomerases Modulate the Timing of Meiotic DNA Breakage and Chromosome Morphogenesis in Saccharomyces cerevisiae

Genetics ◽  
2020 ◽  
Vol 215 (1) ◽  
pp. 59-73 ◽  
Author(s):  
Jonna Heldrich ◽  
Xiaoji Sun ◽  
Luis A. Vale-Silva ◽  
Tovah E. Markowitz ◽  
Andreas Hochwagen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Prophase ◽  
Meiotic Chromosome ◽  
Strand Break ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
Link Type ◽  
Chromosome Synapsis ◽  
Intergenic Regions

During meiotic prophase, concurrent transcription, recombination, and chromosome synapsis place substantial topological strain on chromosomal DNA, but the role of topoisomerases in this context remains poorly defined. Here, we analyzed the roles of topoisomerases I and II (Top1 and Top2) during meiotic prophase in Saccharomyces cerevisiae. We show that both topoisomerases accumulate primarily in promoter-containing intergenic regions of actively transcribing genes, including many meiotic double-strand break (DSB) hotspots. Despite the comparable binding patterns, top1 and top2 mutations have different effects on meiotic recombination. TOP1 disruption delays DSB induction and shortens the window of DSB accumulation by an unknown mechanism. By contrast, temperature-sensitive top2-1 mutants exhibit a marked delay in meiotic chromosome remodeling and elevated DSB signals on synapsed chromosomes. The problems in chromosome remodeling were linked to altered Top2 binding patterns rather than a loss of Top2 catalytic activity, and stemmed from a defect in recruiting the chromosome remodeler Pch2/TRIP13 to synapsed chromosomes. No chromosomal defects were observed in the absence of TOP1. Our results imply independent roles for Top1 and Top2 in modulating meiotic chromosome structure and recombination.

scholarly journals The rec8 gene of Schizosaccharomyces pombe is involved in linear element formation, chromosome pairing and sister-chromatid cohesion during meiosis.

Genetics ◽  
1995 ◽  
Vol 141 (1) ◽  
pp. 61-73
Author(s):  
M Molnar ◽  
J Bähler ◽  
M Sipiczki ◽  
J Kohli
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Chromosome Pairing ◽  
Meiotic Recombination ◽  
Meiotic Prophase ◽  
Meiotic Chromosome ◽  
Linear Element ◽  
Meiotic Chromosomes ◽  
Sister Chromatids ◽  
Linear Elements ◽  
Chromosome Iii

Abstract The fission yeast Schizosaccharomyces pombe does not form tripartite synaptonemal complexes during meiotic prophase, but axial core-like structures (linear elements). To probe the relationship between meiotic recombination and the structure, pairing, and segregation of meiotic chromosomes, we genetically and cytologically characterized the rec8-110 mutant, which is partially deficient in meiotic recombination. The pattern of spore viability indicates that chromosome segregation is affected in the mutant. A detailed segregational analysis in the rec8-110 mutant revealed more spores disomic for chromosome III than in a wild-type strain. Aberrant segregations are caused by precocious segregation of sister chromatids at meiosis I, rather than by nondisjunction as a consequence of lack of crossovers. In situ hybridization further showed that the sister chromatids are separated prematurely during meiotic prophase. Moreover, the mutant forms aberrant linear elements and shows a shortened meiotic prophase. Meiotic chromosome pairing in interstitial and centromeric regions is strongly impaired in rec8-110, whereas the chromosome ends are less deficient in pairing. We propose that the rec8 gene encodes a protein required for linear element formation and that the different phenotypes of rec8-110 reflect direct and indirect consequences of the absence of regular linear elements.

scholarly journals Crossover Interference in Saccharomyces cerevisiae Requires a TID1/RDH54- and DMC1-Dependent Pathway

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1273-1286 ◽  
Author(s):  
Miki Shinohara ◽  
Kazuko Sakai ◽  
Akira Shinohara ◽  
Douglas K Bishop
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Strand Break ◽  
Tetrad Analysis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Crossover Interference ◽  
Meiotic Progression ◽  
Invasion Stage ◽  
Strand Invasion ◽  
Dependent Pathway

Abstract Two RecA-like recombinases, Rad51 and Dmc1, function together during double-strand break (DSB)-mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae. Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors. Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type. Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis. Previous work showed that dmc1Δ mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54. Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Δ cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective. These results support the proposal that crossover interference acts at the strand invasion stage of recombination.

scholarly journals Role of DNA Replication Proteins in Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (16) ◽  
pp. 6891-6899 ◽  
Author(s):  
Xuan Wang ◽  
Grzegorz Ira ◽  
José Antonio Tercero ◽  
Allyson M. Holmes ◽  
John F. X. Diffley ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Synthesis ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
S Phase ◽  
Temperature Sensitive ◽  
Replication Proteins ◽  
Dna Ligases ◽  
Mcm Proteins

ABSTRACT Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4Δ strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase α (Polα), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G2-arrested cells. Whereas PCNA was still essential for MAT switching, neither Polα nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Polα-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

scholarly journals Saccharomyces cerevisiae cdc2 mutants fail to replicate approximately one-third of their nuclear genome.

1983 ◽  
Vol 3 (6) ◽  
pp. 1000-1012 ◽  
Author(s):  
M N Conrad ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Nuclear Genome ◽  
Electron Microscopic Examination ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
Electron Microscopic ◽  
Dna Strands ◽  
Total Dna ◽  
Random Fraction

Chromosomal DNA replication was examined in temperature-sensitive mutants of Saccharomyces cerevisiae defective in a gene required for the completion of S phase at the nonpermissive temperature, 37 degrees C. Based on incorporation of radioactive precursors and density transfer experiments, strains carrying three different alleles of cdc2 failed to replicate approximately one-third of their nuclear genome at 37 degrees C. Whole-cell autoradiography experiments demonstrated that 93 to 96% of the cells synthesized DNA at 37 degrees C. Therefore, all cells failed to replicate part of their genome. DNA isolated from terminally arrested cells was of normal size as measured on neutral and alkaline sucrose gradients, suggesting that partially replicated DNA molecules do not accumulate and that DNA strands are ligated properly in cdc2 mutants. In addition, electron microscopic examination of the equivalent of more than one genome's DNA from arrested cells failed to reveal any partially replicated molecules. The sequences which failed to replicate at 37 degrees C were not highly specific; eight different cloned sequences replicated to the same extent as total DNA. The 2-microns plasmid DNA and rDNA replicated significantly less well than total DNA, but approximately one-half of these sequences replicated at 37 degrees C. These observations suggest that cdc2 mutants are defective in an aspect of initiation of DNA replication common to all chromosomes such that a random fraction of the chromosomes fail to initiate replication at 37 degrees C, but that once initiated, replication proceeds normally.

scholarly journals Stably denatured regions in chromosomal DNA from the cdc2 Saccharomyces cerevisiae cell cycle mutant.

1983 ◽  
Vol 3 (9) ◽  
pp. 1665-1669 ◽  
Author(s):  
M N Conrad ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Early Stage ◽  
Dna Binding Protein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
Cdc2 Gene ◽  
Initiation Of Dna Replication

DNA isolated from Saccharomyces cerevisiae strains carrying temperature-sensitive mutations in the CDC2 gene after incubation at the restrictive temperature contains multiple stably denatured regions 200 to 700 base pairs long. These regions are probably stabilized by a DNA-binding protein. They are found in both replicated and unreplicated portions of DNA molecules, suggesting that they are not an early stage in the initiation of DNA replication.

scholarly journals ATR is required to complete meiotic recombination in mice

2017 ◽  
Author(s):  
Sarai Pacheco ◽  
Andros Maldonado-Linares ◽  
Marina Marcet-Ortega ◽  
Cristina Rojas ◽  
Ana Martínez-Marchal ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Meiotic Prophase ◽  
Protein A ◽  
Homology Search ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Molecular Features ◽  
Chromosome Synapsis ◽  
Meiotic Dsbs

ABSTRACTPrecise execution of recombination during meiosis is essential for forming chromosomally balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Binding of replication protein A (RPA) at resected DSBs fosters association of RAD51 and DMC1, the primary effectors of homology search. It is well appreciated that cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these responses remain poorly understood, particularly in mammals. Here we provide evidence that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.

scholarly journals Dmc1 is a candidate for temperature tolerance during wheat meiosis

2019 ◽  
Author(s):  
Tracie Draeger ◽  
Azahara Martin ◽  
Abdul Kader Alabdullah ◽  
Ali Pendle ◽  
María-Dolores Rey ◽  
...  
Keyword(s):  
High Temperatures ◽  
Chromosome Pairing ◽  
Meiotic Recombination ◽  
Low Temperatures ◽  
Chinese Spring ◽  
Deletion Mutant ◽  
Meiotic Chromosome ◽  
Wheat Chromosome ◽  
Chromosome Synapsis ◽  
Recombination Gene

AbstractWe have assessed the effects of high and low temperatures on meiotic chromosome synapsis and crossover formation in the hexaploid wheat (Triticum aestivum L.) variety ‘Chinese Spring’. At low temperatures, asynapsis and chromosome univalence have been observed before in Chinese Spring lines lacking the long arm of chromosome 5D (5DL), which led to the proposal that 5DL carries a gene (Ltp1) that stabilises wheat chromosome pairing at low temperatures. In the current study, Chinese Spring wild type and 5DL interstitial deletion mutant plants were exposed to low (13°C) or high (30°C) temperatures in controlled environment rooms during a period from premeiotic interphase to early meiosis I. A 5DL deletion mutant was identified whose meiotic chromosomes exhibit extremely high levels of asynapsis and chromosome univalence at metaphase I after seven days at 13°C. This suggests that the mutant, which we name ttmei1 (temperature tolerance in meiosis 1) has a deletion of a gene that, like Ltp1, normally stabilises chromosome pairing at low temperatures. Immunolocalisation of the meiotic proteins ASY1 and ZYP1 on ttmei1 mutants showed that low temperature results in a failure to complete synapsis at pachytene. After 24 hours at 30°C, ttmei1 mutants exhibited a reduced number of crossovers and increased univalence, but to a lesser extent than at 13°C. KASP genotyping revealed that ttmei1 has a 4 Mb deletion in 5DL. Of 41 genes within this deletion region, the strongest candidate for the stabilisation of chromosome pairing at low (and possibly high) temperatures is the meiotic recombination gene Dmc1.Key messageThe meiotic recombination gene Dmc1 on wheat chromosome 5D has been identified as a candidate for the maintenance of normal chromosome synapsis and crossover at low and possibly high temperatures.

scholarly journals Properties of Natural Double-Strand-Break Sites at a Recombination Hotspot in Saccharomyces cerevisiae

Genetics ◽  
2003 ◽  
Vol 165 (1) ◽  
pp. 101-114 ◽  
Author(s):  
Stuart J Haring ◽  
George R Halley ◽  
Alex J Jones ◽  
Robert E Malone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
Meiotic Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
Recombination Hotspot ◽  
Recombination Hotspots ◽  
Naturally Occurring ◽  
High Level ◽  
Site Recognition

Abstract This study addresses three questions about the properties of recombination hotspots in Saccharomyces cerevisiae: How much DNA is required for double-strand-break (DSB) site recognition? Do naturally occurring DSB sites compete with each other in meiotic recombination? What role does the sequence located at the sites of DSBs play? In S. cerevisiae, the HIS2 meiotic recombination hotspot displays a high level of gene conversion, a 3′-to-5′ conversion gradient, and two DSB sites located ∼550 bp apart. Previous studies of hotspots, including HIS2, suggest that global chromosome structure plays a significant role in recombination activity, raising the question of how much DNA is sufficient for hotspot activity. We find that 11.5 kbp of the HIS2 region is sufficient to partially restore gene conversion and both DSBs when moved to another yeast chromosome. Using a variety of different constructs, studies of hotspots have indicated that DSB sites compete with one another for DSB formation. The two naturally occurring DSBs at HIS2 afforded us the opportunity to examine whether or not competition occurs between these native DSB sites. Small deletions of DNA at each DSB site affect only that site; analyses of these deletions show no competition occurring in cis or in trans, indicating that DSB formation at each site at HIS2 is independent. These small deletions significantly affect the frequency of DSB formation at the sites, indicating that the DNA sequence located at a DSB site can play an important role in recombination initiation.

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