scholarly journals Mcmdc2, a minichromosome maintenance (MCM) paralog, is required for repair of meiotic DNA breaks and homolog pairing in mouse meiosis

2016 ◽  
Author(s):  
Adrian J. McNairn ◽  
Vera D. Rinaldi ◽  
John C. Schimenti
Keyword(s):  
Dna Breaks ◽  
Minichromosome Maintenance ◽  
Primordial Follicles ◽  
Homolog Pairing ◽  
Homologous Sequences ◽  
Chromosome Synapsis ◽  
Double Stranded Dna ◽  
Blm Helicase ◽  
Strand Invasion ◽  
Deficient Mice

AbstractThe mammalian Mcm-domain containing 2 (Mcmdc2) gene encodes a protein of unknown function that is homologous to the mini-chromosome maintenance family of DNA replication licensing and helicase factors. Drosophila melanogaster contains two separate genes, the “Mei-MCMs,” that appear to have arisen from a single ancestral Mcmdc2 gene. The Mei-MCMs are involved in promoting meiotic crossovers by blocking the anti-crossover activity of BLM helicase, a function performed by MSH4 and MSH5 in metazoans. Here, we report that MCMDC2-deficient mice of both sexes are viable but sterile. Males fail to produce spermatozoa, and formation of primordial follicles is disrupted in females. Histology and immunocytological analyses of mutant testes revealed that meiosis is arrested in Prophase I, and is characterized by persistent meiotic double-stranded DNA breaks (DSBs), failure of homologous chromosome synapsis and XY body formation, and an absence of crossing over. These phenotypes essentially phenocopy those of MSH4/5 deficient meiocytes. The data indicate that MCMDC2 is essential for invasion of homologous sequences by RAD51- and DMC1-coated ssDNA filaments, or stabilization of recombination intermediates following strand invasion, both of which are needed to drive stable homolog pairing and DSB repair via recombination in mice.

Abnormal rearrangement within the α/δ T-cell receptor locus in lymphomas from Atm-deficient mice

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1940-1946 ◽  
Author(s):  
Marek Liyanage ◽  
Zoë Weaver ◽  
Carrolee Barlow ◽  
Allen Coleman ◽  
Daniel G. Pankratz ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Development ◽  
Cell Receptor ◽  
Chromosome 15 ◽  
Dna Breaks ◽  
Chromosome 14 ◽  
Myc Oncogene ◽  
Double Stranded Dna ◽  
Receptor Locus ◽  
Deficient Mice

Atm-deficient mice (Atm−/−) recapitulate many aspects of the ataxia telangiectasia (AT) syndrome, including the susceptibility to tumors of lymphoid origin. To investigate the mechanism of tumorigenesis, we have examined a panel of 8 thymic lymphomas from Atm−/− mice. AllAtm−/− tumors are of thymic lymphoblastoid origin, display an immature CD3− and CD4+/CD8+ phenotype, and arise coincident with V(D)J recombination. Cytogenetically, all tumors are diploid or near diploid but exhibit multiple chromosome aberrations with an average of 4 abnormal chromosomes per tumor. All the tumors revealed chromosome 14 rearrangements precisely at the T-cell receptorα/δ(Tcrα/δ) locus, suggesting the involvement of V(D)J recombination in these translocations. In addition, 11.5% ofAtm−/− peripheral T cells showed chromosome 14 translocations, suggesting that rearrangements at theTcrα/δ locus occur early during tumor development in the absence of ATM. However, additional genetic aberrations are required for tumorigenesis. For example, translocations involving chromosome 12, often with chromosome 14 (more than 60%), and partial or complete trisomy of chromosome 15, with copy number increases of the c-myc oncogene were frequently observed. These observations suggest that ATM is required for normal rearrangement of the Tcrα/δ locus but not for V(D)J recombination at other loci. The mechanisms that lead to tumorigenesis may be due to the involvement of ATM in monitoring double-stranded DNA breaks.

Abnormal rearrangement within the α/δ T-cell receptor locus in lymphomas from Atm-deficient mice

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1940-1946 ◽  
Author(s):  
Marek Liyanage ◽  
Zoë Weaver ◽  
Carrolee Barlow ◽  
Allen Coleman ◽  
Daniel G. Pankratz ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Development ◽  
Cell Receptor ◽  
Chromosome 15 ◽  
Dna Breaks ◽  
Chromosome 14 ◽  
Myc Oncogene ◽  
Double Stranded Dna ◽  
Receptor Locus ◽  
Deficient Mice

Abstract Atm-deficient mice (Atm−/−) recapitulate many aspects of the ataxia telangiectasia (AT) syndrome, including the susceptibility to tumors of lymphoid origin. To investigate the mechanism of tumorigenesis, we have examined a panel of 8 thymic lymphomas from Atm−/− mice. AllAtm−/− tumors are of thymic lymphoblastoid origin, display an immature CD3− and CD4+/CD8+ phenotype, and arise coincident with V(D)J recombination. Cytogenetically, all tumors are diploid or near diploid but exhibit multiple chromosome aberrations with an average of 4 abnormal chromosomes per tumor. All the tumors revealed chromosome 14 rearrangements precisely at the T-cell receptorα/δ(Tcrα/δ) locus, suggesting the involvement of V(D)J recombination in these translocations. In addition, 11.5% ofAtm−/− peripheral T cells showed chromosome 14 translocations, suggesting that rearrangements at theTcrα/δ locus occur early during tumor development in the absence of ATM. However, additional genetic aberrations are required for tumorigenesis. For example, translocations involving chromosome 12, often with chromosome 14 (more than 60%), and partial or complete trisomy of chromosome 15, with copy number increases of the c-myc oncogene were frequently observed. These observations suggest that ATM is required for normal rearrangement of the Tcrα/δ locus but not for V(D)J recombination at other loci. The mechanisms that lead to tumorigenesis may be due to the involvement of ATM in monitoring double-stranded DNA breaks.

scholarly journals Repair of Meiotic DNA Breaks and Homolog Pairing in Mouse Meiosis Requires a Minichromosome Maintenance (MCM) Paralog

Genetics ◽  
2016 ◽  
Vol 205 (2) ◽  
pp. 529-537 ◽  
Author(s):  
Adrian J. McNairn ◽  
Vera D. Rinaldi ◽  
John C. Schimenti
Keyword(s):  
Dna Breaks ◽  
Minichromosome Maintenance ◽  
Homolog Pairing

scholarly journals DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Prasun Chakraborty ◽  
Kevin Hiom
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Rna Polymerase Ii ◽  
Critical Role ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Recombination Repair ◽  
Dna End Resection ◽  
End Resection ◽  
Rna Polymerase Ii Transcription

AbstractDouble stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

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

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 595-605 ◽  
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.

scholarly journals DNA Binding by the Methanothermobacter thermautotrophicus Cdc6 Protein Is Inhibited by the Minichromosome Maintenance Helicase

2006 ◽  
Vol 188 (12) ◽  
pp. 4577-4580 ◽  
Author(s):  
Rajesh Kasiviswanathan ◽  
Jae-Ho Shin ◽  
Zvi Kelman
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Minichromosome Maintenance ◽  
Single Stranded Dna ◽  
Mutant Proteins ◽  
Dna Binding Activity ◽  
Double Stranded Dna ◽  
Mcm Helicase ◽  
Methanothermobacter Thermautotrophicus

ABSTRACT The Cdc6 proteins from the archaeon Methanothermobacter thermautotrophicus were previously shown to bind double-stranded DNA. It is shown here that the proteins also bind single-stranded DNA. Using minichromosome maintenance (MCM) helicase mutant proteins unable to bind DNA, it was found that the interaction of MCM with Cdc6 inhibits the DNA binding activity of Cdc6.

scholarly journals RAG-Mediated V(D)J Recombination Is Not Essential for Tumorigenesis in Atm-Deficient Mice

2002 ◽  
Vol 22 (9) ◽  
pp. 3174-3177 ◽  
Author(s):  
Lisa K. Petiniot ◽  
Zoë Weaver ◽  
Melanie Vacchio ◽  
Rhuna Shen ◽  
Danny Wangsa ◽  
...  
Keyword(s):  
High Frequency ◽  
Chromosomal Abnormalities ◽  
Chromosomal Translocations ◽  
Thymic Lymphoma ◽  
Double Stranded Dna ◽  
Break Repair ◽  
Rag 1 ◽  
Deficient Mice

ABSTRACT Atm-deficient mice die of malignant thymic lymphomas characterized by translocations within the Tcrα/δ locus, suggesting that tumorigenesis is secondary to aberrant responses to double-stranded DNA (dsDNA) breaks that occur during RAG-dependent V(D)J recombination. We recently demonstrated that development of thymic lymphoma in Atm−/− mice was not prevented by loss of RAG-2. Thymic lymphomas that developed in Rag2−/− Atm−/− mice contained multiple chromosomal abnormalities, but none of these involved the Tcrα/δ locus. These findings indicated that tumorigenesis in Atm−/− mice is mediated by chromosomal translocations secondary to aberrant responses to dsDNA breaks and that V(D)J recombination is an important, but not essential, event in susceptibility. In contrast to these findings, it was recently reported that Rag1−/− Atm−/− mice do not develop thymic lymphomas, a finding that was interpreted as demonstrating a requirement for RAG-dependent recombination in the susceptibility to tumors in Atm-deficient mice. To test the possibility that RAG-1 and RAG-2 differ in their roles in tumorigenesis, we studied Rag1−/− Atm−/− mice in parallel to our previous Rag2−/− Atm−/− study. We found that thymic lymphomas occur at high frequency in Rag1−/− Atm−/− mice and resemble those that occur in Rag2−/− Atm−/− mice. These results indicate that both RAG-1 and RAG-2 are necessary for tumorigenesis involving translocation in the Tcrα/δ locus but that Atm deficiency leads to tumors through a broader RAG-independent predisposition to translocation, related to a generalized defect in dsDNA break repair.

scholarly journals High-resolution mapping of heteroduplex DNA formed during UV-induced and spontaneous mitotic recombination events in yeast

2017 ◽  
Author(s):  
Yi Yin ◽  
Margaret Dominska ◽  
Eunice Yim ◽  
Thomas D. Petes
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Mitotic Recombination ◽  
Template Switching ◽  
Wild Type ◽  
Dna Breaks ◽  
Repair Synthesis ◽  
Heteroduplex Dna ◽  
Dna Molecule ◽  
Double Stranded Dna

AbstractDouble-stranded DNA breaks (DSBs) can be generated by both endogenous and exogenous agents. In diploid yeast strains, such breaks are usually repaired by homologous recombination (HR), and a number of different HR pathways have been described. An early step for all HR pathways is formation of a heteroduplex, in which a single-strand from the broken DNA molecule pairs with a strand derived from an intact DNA molecule. If the two strands of DNA are not identical, within the heteroduplex DNA (hetDNA), there will be mismatches. In a wild-type strain, these mismatches are removed by the mismatch repair (MMR) system. In strains lacking MMR, the mismatches persist and can be detected by a variety of genetic and physical techniques. Most previous studies involving hetDNA formed during mitotic recombination have been restricted to a single locus with DSBs induced at a defined position by a site-specific endonuclease. In addition, in most of these studies, recombination between repeated genes was examined; in such studies, the sequence homologies were usually less than 5 kb. In the present study, we present a global mapping of hetDNA formed in a UV-treated MMR-defective mlh1 strain. Although about two-thirds of the recombination events were associated with hetDNA with a continuous array of unrepaired mismatches, in about one-third of the events, we found regions of unrepaired mismatches flanking regions without mismatches. We suggest that these discontinuous hetDNAs involve template switching during repair synthesis, repair of a double-stranded DNA gap, and/or Mlh1-independent MMR. Many of our observed events are not explicable by the simplest form of the double-strand break repair (DSBR) model of recombination. We also studied hetDNA associated with spontaneous recombination events selected on chromosomes IV and V in a wild-type strain. The interval on chromosome IV contained a hotspot for spontaneous crossovers generated by an inverted pair of transposable elements (HS4). We showed that HS4-induced recombination events are associated with the formation of very large (>30 kb) double-stranded DNA gaps.

scholarly journals CRISPR enriches for cells with mutations in a p53-related interactome, and this can be inhibited

2021 ◽  
Author(s):  
Long Jiang ◽  
Katrine Ingelshed ◽  
Yunbing Shen ◽  
Sanjaykumar V. Boddul ◽  
Vaishnavi Srinivasan Iyer ◽  
...  
Keyword(s):  
Cellular Response ◽  
Human Cancer ◽  
Genetic Alterations ◽  
Dna Breaks ◽  
Data Set ◽  
Human Cancer Cell Lines ◽  
Cycle Arrest ◽  
Double Stranded Dna ◽  
A Cell

CRISPR/Cas9 can be used to inactivate or modify genes by inducing double-stranded DNA breaks1–3. As a protective cellular response, DNA breaks result in p53-mediated cell cycle arrest and activation of cell death programs4,5. Inactivating p53 mutations are the most commonly found genetic alterations in cancer, highlighting the important role of the gene6–8. Here, we show that cells deficient in p53, as well as in genes of a core CRISPR-p53 tumor suppressor interactome, are enriched in a cell population when CRISPR is applied. Such enrichment could pose a challenge for clinical CRISPR use. Importantly, we identify that transient p53 inhibition suppresses the enrichment of cells with these mutations. Furthermore, in a data set of >800 human cancer cell lines, we identify parameters influencing the enrichment of p53 mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR-p53 axis. Taken together, our data identify strategies enabling safe CRISPR use.

scholarly journals Mechanism for nuclease regulation in RecBCD

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Martin Wilkinson ◽  
Yuriy Chaban ◽  
Dale B Wigley
Keyword(s):  
Nuclease Activity ◽  
Binding Mode ◽  
Sh3 Domain ◽  
Bacterial Cells ◽  
Dna Breaks ◽  
Nuclease Domain ◽  
Double Stranded Dna ◽  
Large Conformational Change ◽  
Enzyme Complexes ◽  
Dna Substrate

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 Å resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5’-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains and, to our knowledge, this is the first example of peptide-binding of an SH3 domain in a bacterial system.

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