scholarly journals Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis

2019 ◽  
Author(s):  
Hiroyuki Sasanuma ◽  
Hana Subhan M. Sakurai ◽  
Yuko Furihata ◽  
Kiran Challa ◽  
Lira Palmer ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromosome Segregation ◽  
Dna Recombination ◽  
Dna Helicase ◽  
Aggregate Formation ◽  
Late Prophase ◽  
Prophase I ◽  
Early Protein ◽  
Srs2 Helicase ◽  
Yeast Meiosis

AbstractProper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase-I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci or among the foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphase-I and -II, suggesting the absence of DNA damage-induced cell-cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase-I.

scholarly journals Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis

Chromosoma ◽  
2019 ◽  
Vol 128 (3) ◽  
pp. 453-471 ◽  
Author(s):  
Hiroyuki Sasanuma ◽  
Hana Subhan M. Sakurai ◽  
Yuko Furihata ◽  
Kiran Challa ◽  
Lira Palmer ◽  
...  
Keyword(s):  
Dna Damage ◽  
Late Prophase ◽  
Prophase I ◽  
Srs2 Helicase ◽  
Yeast Meiosis

scholarly journals The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Alex Bronstein ◽  
Lihi Gershon ◽  
Gilad Grinberg ◽  
Elisa Alonso-Perez ◽  
Martin Kupiec
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
Dna Helicase ◽  
Main Role ◽  
Helicase Domain ◽  
Helicase Activity ◽  
C Terminus ◽  
Srs2 Helicase

ABSTRACTHomologous recombination (HR) is a mechanism that repairs a variety of DNA lesions. Under certain circumstances, however, HR can generate intermediates that can interfere with other cellular processes such as DNA transcription or replication. Cells have therefore developed pathways that abolish undesirable HR intermediates. TheSaccharomyces cerevisiaeyeast Srs2 helicase has a major role in one of these pathways. Srs2 also works during DNA replication and interacts with the clamp PCNA. The relative importance of Srs2’s helicase activity, Rad51 removal function, and PCNA interaction in genome stability remains unclear. We created a newSRS2allele [srs2(1-850)] that lacks the whole C terminus, containing the interaction site for Rad51 and PCNA and interactions with many other proteins. Thus, the new allele encodes an Srs2 protein bearing only the activity of the DNA helicase. We find that the interactions of Srs2 with Rad51 and PCNA are dispensable for the main role of Srs2 in the repair of DNA damage in vegetative cells and for proper completion of meiosis. On the other hand, it has been shown that in cells impaired for the DNA damage tolerance (DDT) pathways, Srs2 generates toxic intermediates that lead to DNA damage sensitivity; we show that this negative Srs2 activity requires the C terminus of Srs2. Dissection of the genetic interactions of thesrs2(1-850) allele suggest a role for Srs2’s helicase activity in sister chromatid cohesion. Our results also indicate that Srs2’s function becomes more central in diploid cells.IMPORTANCEHomologous recombination (HR) is a key mechanism that repairs damaged DNA. However, this process has to be tightly regulated; failure to regulate it can lead to genome instability. The Srs2 helicase is considered a regulator of HR; it was shown to be able to evict the recombinase Rad51 from DNA. Cells lacking Srs2 exhibit sensitivity to DNA-damaging agents, and in some cases, they display defects in DNA replication. The relative roles of the helicase and Rad51 removal activities of Srs2 in genome stability remain unclear. To address this question, we created a new Srs2 mutant which has only the DNA helicase domain. Our study shows that only the DNA helicase domain is needed to deal with DNA damage and assist in DNA replication during vegetative growth and in meiosis. Thus, our findings shift the view on the role of Srs2 in the maintenance of genome integrity.

scholarly journals HIM-17 regulates the position of recombination events and GSP-1/2 localization to establish short arm identity on bivalents in meiosis

2021 ◽  
Vol 118 (17) ◽  
pp. e2016363118
Author(s):  
Saravanapriah Nadarajan ◽  
Elisabeth Altendorfer ◽  
Takamune T. Saito ◽  
Marina Martinez-Garcia ◽  
Monica P. Colaiácovo
Keyword(s):  
Chromosome Segregation ◽  
Normal Chromosome ◽  
Early Prophase ◽  
Dna Double Strand Breaks ◽  
Late Prophase ◽  
Strand Breaks ◽  
Meiotic Progression ◽  
Prophase I ◽  
Chromatid Cohesion ◽  
Normal Expression

The position of recombination events established along chromosomes in early prophase I and the chromosome remodeling that takes place in late prophase I are intrinsically linked steps of meiosis that need to be tightly regulated to ensure accurate chromosome segregation and haploid gamete formation. Here, we show that RAD-51 foci, which form at the sites of programmed meiotic DNA double-strand breaks (DSBs), exhibit a biased distribution toward off-centered positions along the chromosomes in wild-type Caenorhabditis elegans, and we identify two meiotic roles for chromatin-associated protein HIM-17 that ensure normal chromosome remodeling in late prophase I. During early prophase I, HIM-17 regulates the distribution of DSB-dependent RAD-51 foci and crossovers on chromosomes, which is critical for the formation of distinct chromosome subdomains (short and long arms of the bivalents) later during chromosome remodeling. During late prophase I, HIM-17 promotes the normal expression and localization of protein phosphatases GSP-1/2 to the surface of the bivalent chromosomes and may promote GSP-1 phosphorylation, thereby antagonizing Aurora B kinase AIR-2 loading on the long arms and preventing premature loss of sister chromatid cohesion. We propose that HIM-17 plays distinct roles at different stages during meiotic progression that converge to promote normal chromosome remodeling and accurate chromosome segregation.

scholarly journals Srs2 and Sgs1 DNA Helicases Associate with Mre11 in Different Subcomplexes following Checkpoint Activation and CDK1-Mediated Srs2 Phosphorylation

2005 ◽  
Vol 25 (13) ◽  
pp. 5738-5751 ◽  
Author(s):  
Irene Chiolo ◽  
Walter Carotenuto ◽  
Giulio Maffioletti ◽  
John H. J. Petrini ◽  
Marco Foiani ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Genome Instability ◽  
Dna Recombination ◽  
Dna Helicase ◽  
Checkpoint Activation ◽  
Dna Helicases ◽  
Checkpoint Response ◽  
Checkpoint Kinases ◽  
Genes Encoding

ABSTRACT Mutations in the genes encoding the BLM and WRN RecQ DNA helicases and the MRE11-RAD50-NBS1 complex lead to genome instability and cancer predisposition syndromes. The Saccharomyces cerevisiae Sgs1 RecQ helicase and the Mre11 protein, together with the Srs2 DNA helicase, prevent chromosome rearrangements and are implicated in the DNA damage checkpoint response and in DNA recombination. By searching for Srs2 physical interactors, we have identified Sgs1 and Mre11. We show that Srs2, Sgs1, and Mre11 form a large complex, likely together with yet unidentified proteins. This complex reorganizes into Srs2-Mre11 and Sgs1-Mre11 subcomplexes following DNA damage-induced activation of the Mec1 and Tel1 checkpoint kinases. The defects in subcomplex formation observed in mec1 and tel1 cells can be recapitulated in srs2-7AV mutants that are hypersensitive to intra-S DNA damage and are altered in the DNA damage-induced and Cdk1-dependent phosphorylation of Srs2. Altogether our observations indicate that Mec1- and Tel1-dependent checkpoint pathways modulate the functional interactions between Srs2, Sgs1, and Mre11 and that the Srs2 DNA helicase represents an important target of the Cdk1-mediated cellular response induced by DNA damage.

scholarly journals Meiosis-specific prophase-like pathway controls cleavage-independent release of cohesin by Wapl phosphorylation

2018 ◽  
Author(s):  
Kiran Challa ◽  
V Ghanim Fajish ◽  
Miki Shinohara ◽  
Franz Klein ◽  
Susan M. Gasser ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Meiotic Prophase ◽  
Budding Yeast ◽  
Cell Cycle Regulators ◽  
Late Prophase ◽  
Homologous Chromosomes ◽  
Prophase I ◽  
Meiotic Prophase I ◽  
Meiotic Chromosomes

AbstractSister chromatid cohesion on chromosome arms is essential for the segregation of homologous chromosomes during meiosis I while it is dispensable for sister chromatid separation during mitosis. It was assumed that, unlike the situation in mitosis, chromosome arms retain cohesion prior to onset of anaphase-I. Paradoxically, reduced immunostaining signals of meiosis-specific cohesin, including the kleisin Rec8, from the chromosomes were observed during late prophase-I of budding yeast. This decrease is seen in the absence of Rec8 cleavage and depends on condensin-mediated recruitment of Polo-like kinase (PLK/Cdc5). In this study, we confirmed that this release indeed accompanies the dissociation of acetylated Smc3 as well as Rec8 from meiotic chromosomes during late prophase-I. This release requires, in addition to PLK, the cohesin regulator, Wapl (Rad61/Wpl1 in yeast), and Dbf4-dependent Cdc7 kinase (DDK). Meiosis-specific phosphorylation of Rad61/Wpl1 and Rec8 by PLK and DDK collaboratively promote this release. This process is similar to the vertebrate “prophase” pathway for cohesin release during G2 phase and pro-metaphase. In yeast, meiotic cohesin release coincides with PLK-dependent compaction of chromosomes in late meiotic prophase-I. We suggest that yeast uses this highly regulated cleavage-independent pathway to remove cohesin during late prophase-I to facilitate morphogenesis of condensed metaphase-I chromosomes.Author SummaryIn meiosis the life and health of future generations is decided upon. Any failure in chromosome segregation has a detrimental impact. Therefore, it is currently believed that the physical connections between homologous chromosomes are maintained by meiotic cohesin with exceptional stability. Indeed, it was shown that cohesive cohesin does not show an appreciable turnover during long periods in oocyte development. In this context, it was long assumed but not properly investigated, that the prophase pathway for cohesin release would be specific to mitotic cells and will be safely suppressed during meiosis so as not to endanger the valuable chromosome connections. However, a previous study on budding yeast meiosis suggests the presence of cleavage-independent pathway of cohesin release during late prophase-I. In the work presented here we confirmed that the prophase pathway is not suppressed during meiosis, at least in budding yeast and showed that this cleavage-independent release is regulated by meiosis-specific phosphorylation of two cohesin subunits, Rec8 and Rad61(Wapl) by two cell-cycle regulators, PLK and DDK. Our results suggest that late meiotic prophase-I actively controls cohesin dynamics on meiotic chromosomes for chromosome segregation.

scholarly journals The Srs2 helicase dampens DNA damage checkpoint by recycling RPA from chromatin

2021 ◽  
Vol 118 (8) ◽  
pp. e2020185118
Author(s):  
Nalini Dhingra ◽  
Sahiti Kuppa ◽  
Lei Wei ◽  
Nilisha Pokhrel ◽  
Silva Baburyan ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genotoxic Stress ◽  
Dna Helicase ◽  
Dna Damage Checkpoint ◽  
Recombinational Repair ◽  
Checkpoint Signaling ◽  
Cellular Survival ◽  
Moderate Reduction ◽  
Srs2 Helicase ◽  
Harmful Effects

The DNA damage checkpoint induces many cellular changes to cope with genotoxic stress. However, persistent checkpoint signaling can be detrimental to growth partly due to blockage of cell cycle resumption. Checkpoint dampening is essential to counter such harmful effects, but its mechanisms remain to be understood. Here, we show that the DNA helicase Srs2 removes a key checkpoint sensor complex, RPA, from chromatin to down-regulate checkpoint signaling in budding yeast. The Srs2 and RPA antagonism is supported by their numerous suppressive genetic interactions. Importantly, moderate reduction of RPA binding to single-strand DNA (ssDNA) rescues hypercheckpoint signaling caused by the loss of Srs2 or its helicase activity. This rescue correlates with a reduction in the accumulated RPA and the associated checkpoint kinase on chromatin insrs2mutants. Moreover, our data suggest that Srs2 regulation of RPA is separable from its roles in recombinational repair and critically contributes to genotoxin resistance. We conclude that dampening checkpoint by Srs2-mediated RPA recycling from chromatin aids cellular survival of genotoxic stress and has potential implications in other types of DNA transactions.

scholarly journals The conserved Cdc14 phosphatase ensures effective resolution of meiotic recombination intermediates

2019 ◽  
Author(s):  
Paula Alonso-Ramos ◽  
David Álvarez-Melo ◽  
Katerina Strouhalova ◽  
Carolina Pascual-Silva ◽  
George B. Garside ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Meiotic Recombination ◽  
Meiotic Division ◽  
Budding Yeast ◽  
Direct Role ◽  
Independent Manner ◽  
Prophase I ◽  
New Findings ◽  
Yeast Meiosis

AbstractMeiotic defects derived from incorrect DNA repair during gametogenesis can lead to mutations, aneuploidies and infertility. Effective and coordinated resolution of meiotic recombination intermediates is necessary to accomplish both rounds of successful chromosome segregation. Cdc14 is an evolutionarily conserved dual-specificity phosphatase required for mitotic exit and meiotic progression. Mutations that inactivate the phosphatase lead to meiotic failure. Here, we have identified previously undescribed roles of Cdc14 in ensuring correct meiotic recombination. We found that recombination intermediates accumulate during prophase I when Cdc14 is depleted. Furthermore, Cdc14 plays a role in correct homolog disjunction at the end of anaphase I, both by modulating the timely removal of arm-cohesion between sister chromatids and by promoting elimination of SPO11-dependent entanglements. We also demonstrate that Cdc14 is required for correct sister chromatid segregation during the second meiotic division, independent of centromeric cohesion but dependent on the correct reduplication of SPBs during meiosis II, and on the activity of the Holliday Junction resolvase Yen1/GEN1. Timely activation of Yen1/GEN1 in anaphase I and II is impaired in the meiosis defective allele, cdc143HA. Based on these new findings, we propose previously undescribed functions of Cdc14 in the regulation of meiotic recombination; roles that are independent of sister chromatid cohesion, spindle dynamics and the metabolism of gamete morphogenesis.Author SummaryMeiotic recombination is fundamental for sexual reproduction, with efficient and orchestrated resolution of recombination intermediates critical for correct chromosome segregation. Homologous recombination is initiated by the introduction of programmed DNA Double-Strand Breakds (DSBs) followed by the formation of complex branched DNA intermediates, including double Holliday Junctions (dHJs). These recombination intermediates are eventually repaired into crossover or non-crossover products. In some cases, unresolved recombination intermediates, or toxic repair products, might persist until the metaphase to anaphase transition, requiring a set of late-acting repair enzymes to process them. Unrestrained activity of these enzymes, however, is equally detrimental for genome integrity, thus several layers of regulation tightly control them. For example, in budding yeast meiosis, Yen1/GEN1 is mainly activated during the second meiotic division, although how it is activated is unknown. Here, we have identified that the phosphatase Cdc14 is required during meiotic divisions for timely nuclear localization and activation of Yen1 in budding yeast meiosis. Additionally, we have been able to identify previously undescribed roles of Cdc14 in controlling meiotic recombination. Strikingly, we found that levels of recombination intermediates increase during prophase I in cdc14 meiotic deficient cells, indicating that Cdc14 plays a direct role in monitoring meiotic DSB repair, possibly in Yen1-independent manner. Resolution of recombination intermediates in the absence of Cdc14 is dependent on SGS1 and MUS81/MMS4, otherwise accumulating different types of aberrant recombination intermediates and a highly reduced efficiency in CO formation. Deficient resolution of JMs in cdc14 meiotic cells, together with difficulties in SPB reduplication, likely contribute to the missegregation problems observed during the second meiotic division.

Premature ovarian ageing following heterozygous loss of Senataxin

2020 ◽  
Author(s):  
G N Subramanian ◽  
M Lavin ◽  
H A Homer
Keyword(s):  
Dna Damage ◽  
Neurodegenerative Disorder ◽  
Dna Helicase ◽  
Dna Integrity ◽  
Ovarian Activity ◽  
Homozygous Mutation ◽  
Female Mice ◽  
Mating Trial ◽  
High Prevalence

Abstract Premature loss of ovarian activity before 40 years of age is known as primary ovarian insufficiency (POI) and occurs in ∼1% of women. A more subtle decline in ovarian activity, known as premature ovarian ageing (POA), occurs in ∼10% of women. Despite the high prevalence of POA, very little is known regarding its genetic causation. Senataxin (SETX) is an RNA/DNA helicase involved in repair of oxidative stress-induced DNA damage. Homozygous mutation of SETX leads to the neurodegenerative disorder, ataxia oculomotor apraxia type 2 (AOA2). There have been reports of POI in AOA2 females suggesting a link between SETX and ovarian ageing. Here, we studied female mice lacking either one (Setx+/−) or both (Setx−/−) copies of SETX over a 12- to 14-month period. We find that DNA damage is increased in oocytes from 8-month-old Setx+/− and Setx−/− females compared with Setx+/+ oocytes leading to a marked reduction in all classes of ovarian follicles at least 4 months earlier than typically occurs in female mice. Furthermore, during a 12-month long mating trial, Setx+/− and Setx−/− females produced significantly fewer pups than Setx+/+ females from 7 months of age onwards. These data show that SETX is critical for preventing POA in mice, likely by preserving DNA integrity in oocytes. Intriguingly, heterozygous Setx loss causes an equally severe impact on ovarian ageing as homozygous Setx loss. Because heterozygous SETX disruption is less likely to produce systemic effects, SETX compromise could underpin some cases of insidious POA.

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

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