scholarly journals REC114 partner ANKRD31 controls number, timing and location of meiotic DNA breaks

2018 ◽  
Author(s):  
Michiel Boekhout ◽  
Mehmet E. Karasu ◽  
Juncheng Wang ◽  
Laurent Acquaviva ◽  
Florencia Pratto ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Homologous Recombination ◽  
Meiotic Chromosome ◽  
Double Strand Breaks ◽  
Pleckstrin Homology Domain ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Pseudoautosomal Regions ◽  
Genomic Locations

Double-strand breaks (DSBs) initiate the homologous recombination that is crucial for meiotic chromosome pairing and segregation. Here we unveil mouse ANKRD31 as a lynchpin governing multiple aspects of DSB formation. Spermatocytes lacking ANKRD31 have altered DSB locations and fail to target DSBs to sex chromosomes’ pseudoautosomal regions (PAR). They also have delayed/fewer recombination sites but, paradoxically, more DSBs, suggesting DSB dysregulation. Unrepaired DSBs and pairing failures—stochastic on autosomes, nearly absolute on X and Y—cause meiotic arrest and sterility in males. Ankrd31-deficient females have reduced oocyte reserves. A crystal structure defines direct ANKRD31– REC114 molecular contacts and reveals a surprising pleckstrin homology domain in REC114. In vivo, ANKRD31 stabilizes REC114 association with the PAR and elsewhere. Our findings inform a model that ANKRD31 is a scaffold anchoring REC114 and other factors to specific genomic locations, promoting efficient and timely DSB formation but possibly also suppressing formation of clustered DSBs.

scholarly journals Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Sucheta Arora ◽  
Rajashree A. Deshpande ◽  
Martin Budd ◽  
Judy Campbell ◽  
America Revere ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

scholarly journals Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.

1997 ◽  
Vol 17 (11) ◽  
pp. 6386-6393 ◽  
Author(s):  
D G Taghian ◽  
J A Nickoloff
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Double Strand Breaks ◽  
Chromosomal Dna ◽  
Exogenous Dna ◽  
Strand Breaks ◽  
Conversion Tract

Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.

scholarly journals Long Palindromic Sequences Induce Double-Strand Breaks during Meiosis in Yeast

2000 ◽  
Vol 20 (10) ◽  
pp. 3449-3458 ◽  
Author(s):  
Farooq Nasar ◽  
Craig Jankowski ◽  
Dilip K. Nag
Keyword(s):  
Homologous Recombination ◽  
Genomic Instability ◽  
Double Strand Breaks ◽  
Gene Products ◽  
Strand Breaks ◽  
Hairpin Formation ◽  
Palindromic Sequences ◽  
Normal Meiosis ◽  
Eukaryotic Genomes

ABSTRACT Inverted-repeated or palindromic sequences have been found to occur in both prokaryotic and eukaryotic genomes. Such repeated sequences are usually short and present at several functionally important regions in the genome. However, long palindromic sequences are rare and are a major source of genomic instability. The palindrome-mediated genomic instability is believed to be due to cruciform or hairpin formation and subsequent cleavage of this structure by structure-specific nucleases. Here we present both genetic and physical evidence that long palindromic sequences (>50 bp) generate double-strand breaks (DSBs) at a high frequency during meiosis in the yeast Saccharomyces cerevisiae. The palindrome-mediated DSB formation depends on the primary sequence of the inverted repeat and the location and length of the repeated units. The DSB formation at the palindrome requires all of the gene products that are known to be responsible for DSB formation at the normal meiosis-specific sites. Since DSBs are initiators of nearly all meiotic recombination events, most of the palindrome-induced breaks appear to be repaired by homologous recombination. Our results suggest that short palindromic sequences are highly stable in vivo. In contrast, long palindromic sequences make the genome unstable by inducing DSBs and such sequences are usually removed from the genome by homologous recombination events.

scholarly journals Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination

2021 ◽  
Vol 118 (11) ◽  
pp. e2021963118
Author(s):  
Donna R. Whelan ◽  
Eli Rothenberg
Keyword(s):  
Homologous Recombination ◽  
Single Molecule ◽  
Protein A ◽  
Super Resolution ◽  
Initial Step ◽  
Double Strand Breaks ◽  
Homology Search ◽  
Major Pathway ◽  
Strand Breaks

Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSBs). The initial step that drives the HR process is resection of DNA at the DSB, during which a multitude of nucleases, mediators, and signaling proteins accumulates at the damage foci in a manner that remains elusive. Using single-molecule localization super-resolution (SR) imaging assays, we specifically visualize the spatiotemporal behavior of key mediator and nuclease proteins as they resect DNA at single-ended double-strand breaks (seDSBs) formed at collapsed replication forks. By characterizing these associations, we reveal the in vivo dynamics of resection complexes involved in generating the long single-stranded DNA (ssDNA) overhang prior to homology search. We show that 53BP1, a protein known to antagonize HR, is recruited to seDSB foci during early resection but is spatially separated from repair activities. Contemporaneously, CtBP-interacting protein (CtIP) and MRN (MRE11-RAD51-NBS1) associate with seDSBs, interacting with each other and BRCA1. The HR nucleases EXO1 and DNA2 are also recruited and colocalize with each other and with the repair helicase Bloom syndrome protein (BLM), demonstrating multiple simultaneous resection events. Quantification of replication protein A (RPA) accumulation and ssDNA generation shows that resection is completed 2 to 4 h after break induction. However, both BRCA1 and BLM persist later into HR, demonstrating potential roles in homology search and repair resolution. Furthermore, we show that initial recruitment of BRCA1 and removal of Ku are largely independent of MRE11 exonuclease activity but dependent on MRE11 endonuclease activity. Combined, our observations provide a detailed description of resection during HR repair.

scholarly journals Stepwise 5′ DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble

2019 ◽  
Vol 116 (12) ◽  
pp. 5505-5513 ◽  
Author(s):  
Elda Cannavo ◽  
Giordano Reginato ◽  
Petr Cejka
Keyword(s):  
Atp Hydrolysis ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Dna Strands ◽  
Dna Strand ◽  
The Individual

To repair DNA double-strand breaks by homologous recombination, the 5′-terminated DNA strands must first be resected to produce 3′ overhangs. Mre11 fromSaccharomyces cerevisiaeis a 3′ → 5′ exonuclease that is responsible for 5′ end degradation in vivo. Using plasmid-length DNA substrates and purified recombinant proteins, we show that the combined exonuclease and endonuclease activities of recombinant MRX-Sae2 preferentially degrade the 5′-terminated DNA strand, which extends beyond the vicinity of the DNA end. Mechanistically, Rad50 restricts the Mre11 exonuclease in an ATP binding-dependent manner, preventing 3′ end degradation. Phosphorylated Sae2, along with stimulating the MRX endonuclease as shown previously, also overcomes this inhibition to promote the 3′ → 5′ exonuclease of MRX, which requires ATP hydrolysis by Rad50. Our results support a model in which MRX-Sae2 catalyzes 5′-DNA end degradation by stepwise endonucleolytic DNA incisions, followed by exonucleolytic 3′ → 5′ degradation of the individual DNA fragments. This model explains how both exonuclease and endonuclease activities of Mre11 functionally integrate within the MRX-Sae2 ensemble to resect 5′-terminated DNA.

scholarly journals The Anaphase-Promoting Complex/Cyclosome Controls Repair and Recombination by Ubiquitylating Rhp54 in Fission Yeast

2008 ◽  
Vol 28 (12) ◽  
pp. 3905-3916 ◽  
Author(s):  
Michelle Trickey ◽  
Margaret Grimaldi ◽  
Hiroyuki Yamano
Keyword(s):  
Homologous Recombination ◽  
Fission Yeast ◽  
Meiotic Chromosome ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Anaphase Promoting Complex ◽  
Strand Breaks ◽  
Dna Damaging Agents ◽  
Diploid Cells ◽  
Sister Chromatid Recombination

ABSTRACT Homologous recombination (HR) is important for maintaining genome integrity and for the process of meiotic chromosome segregation and the generation of variation. HR is regulated throughout the cell cycle, being prevalent in the S and G2 phases and suppressed in the G1 phase. Here we show that the anaphase-promoting complex/cyclosome (APC/C) regulates homologous recombination in the fission yeast Schizosaccharomyces pombe by ubiquitylating Rhp54 (an ortholog of Rad54). We show that Rhp54 is a novel APC/C substrate that is destroyed in G1 phase in a KEN-box- and Ste9/Fizzy-related manner. The biological consequences of failing to temporally regulate HR via Rhp54 degradation are seen in haploid cells only in the absence of antirecombinase Srs2 function and are more extensive in diploid cells, which become sensitive to a range of DNA-damaging agents, including hydroxyurea, methyl methanesulfonate, bleomycin, and UV. During meiosis, expression of nondegradable Rhp54 inhibits interhomolog recombination and stimulates sister chromatid recombination. We thus propose that it is critical to control levels of Rhp54 in G1 to suppress HR repair of double-strand breaks and during meiosis to coordinate interhomolog recombination.

scholarly journals ERK phosphorylates chromosomal axis component HORMA domain protein HTP-1 to regulate oocyte numbers

2020 ◽  
Vol 6 (44) ◽  
pp. eabc5580
Author(s):  
Debabrata Das ◽  
Shin-Yu Chen ◽  
Swathi Arur
Keyword(s):  
Meiotic Chromosome ◽  
Reproductive Fitness ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Critical Determinant ◽  
Strand Breaks ◽  
Oocyte Number ◽  
Meiotic Progression ◽  
Complex Extension

Oocyte numbers, a critical determinant of female reproductive fitness, are highly regulated, yet the mechanisms underlying this regulation remain largely undefined. In the Caenorhabditis elegans gonad, RAS/extracellular signal–regulated kinase (ERK) signaling regulates oocyte numbers; mechanisms are unknown. We show that the RAS/ERK pathway phosphorylates meiotic chromosome axis protein HTP-1 at serine-325 to control chromosome dynamics and regulate oocyte number. Phosphorylated HTP-1(S325) accumulates in vivo in an ERK-dependent manner in early-mid pachytene stage germ cells and is necessary for synaptonemal complex extension and/or maintenance. Lack of HTP-1 phosphorylation leads to asynapsis and persistence of meiotic double-strand breaks, causing delayed meiotic progression and reduced oocyte number. In contrast, early onset of ERK activation causes precocious meiotic progression, resulting in increased oocyte number, which is reversed by removal of HTP-1 phosphorylation. The RAS/ERK/HTP-1 signaling cascade thus functions to monitor formation and maintenance of synapsis for timely resolution of double-strand breaks, oocyte production, and reproductive fitness.

scholarly journals DNA-RNA hybrids at DSBs interfere with repair by homologous recombination

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Pedro Ortega ◽  
Jose Antonio Mérida-Cerro ◽  
Ana G Rondón ◽  
Belén Gómez-González ◽  
Andrés Aguilera
Keyword(s):  
Homologous Recombination ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Positive Role ◽  
Strand Breaks ◽  
Endonucleolytic Cleavage ◽  
Chromosomal Recombination

DNA double strand breaks (DSBs) are the most harmful DNA lesions and their repair is crucial for cell viability and genome integrity. The readout of DSB repair may depend on whether DSBs occur at transcribed versus non-transcribed regions. Some studies have postulated that DNA-RNA hybrids form at DSBs to promote recombinational repair, but others have challenged this notion. To directly assess whether hybrids formed at DSBs promote or interfere with recombinational repair we have used plasmid and chromosomal-based systems for the analysis of DSB-induced recombination in Saccharomyces cerevisiae. We show that, as expected, DNA-RNA hybrid formation is stimulated at DSBs. In addition, mutations that promote DNA-RNA hybrid accumulation, such as hpr1∆ and rnh1∆ rnh201∆, cause high levels of plasmid loss when DNA breaks are induced at sites that are transcribed. Importantly, we show that high levels or unresolved DNA-RNA hybrids at the breaks interfere with their repair by homologous recombination. This interference is observed for both plasmid and chromosomal recombination and is independent of whether the DSB is generated by endonucleolytic cleavage or by DNA replication. These data support a model in which DNA-RNA hybrids form fortuitously at DNA breaks during transcription, and need to be removed to allow recombinational repair, rather than playing a positive role.

scholarly journals Homologous recombination as a resistance mechanism to replication-induced double-strand breaks caused by the antileukemia agent CNDAC

Blood ◽  
2010 ◽  
Vol 116 (10) ◽  
pp. 1737-1746 ◽  
Author(s):  
Xiaojun Liu ◽  
Yaqing Wang ◽  
Sherri Benaissa ◽  
Akira Matsuda ◽  
Hagop Kantarjian ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cell Survival ◽  
Resistance Mechanism ◽  
Double Strand Breaks ◽  
Single Strand ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Single Strand Break ◽  
Strand Breaks

Abstract The nucleoside analog 2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosyl-cytosine (CNDAC), currently in clinical trials for hematologic malignancies, has a novel action mechanism of causing a single-strand break after its incorporation into DNA. Double-strand breaks (DSBs) are generated thereafter in vivo and, if not repaired, pose lethal impact on cell survival. This study sought to define the mechanisms by which CNDAC-induced DSBs are formed and repaired. We demonstrated that single-strand breaks induced by CNDAC incorporation into DNA were converted to DSBs when cells progressed into the subsequent S-phase. CNDAC-induced DSBs were products of replication, rather than a consequence of apoptosis. ATM, the activator of homologous recombination (HR), was essential for cell survival after CNDAC treatment in cell lines and in primary acute myeloid leukemia samples, as were the HR components, Rad51, Xrcc3, and Brca2. Furthermore, formation of sister chromatid exchanges, a hallmark of HR, increased significantly after CNDAC-treated cells had progressed into a second replication cycle. In contrast, neither the replication stress sensor ATR nor DNA-PK, the initiator of nonhomologous end-joining of DSB, was involved in repair of CNDAC-induced damage. Together, these results indicate that HR, but not nonhomologous end-joining, is the major repair or survival mechanism for DNA damage caused by CNDAC.

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