scholarly journals Chromosome-wide distribution and characterization of intersubspecific meiotic noncrossovers in mice

2019 ◽  
Author(s):  
Vaclav Gergelits ◽  
Emil Parvanov ◽  
Petr Simecek ◽  
Jiri Forejt
Keyword(s):  
Mouse Chromosome ◽  
Hybrid Sterility ◽  
Wide Distribution ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Dsbs ◽  
Significant Deficit ◽  
Chromosome Substitution Strains

AbstractDuring meiosis, the recombination-initiating DNA double-strand breaks (DSBs) are repaired by crossovers or noncrossovers (gene conversions). While crossovers are easily detectable, the noncrossover identification is hampered by the small size of their converted tracts and the necessity of sequence polymorphism to detect them. We report identification and characterization of a mouse chromosome-wide set of noncrossovers by NGS sequencing of 10 mouse chromosome substitution strains. Based on 94 identified noncrossovers we determined the mean length of a conversion tract to be 32 basepairs. The spatial chromosome-wide distribution of noncrossovers and crossovers significantly differed, though both sets overlapped the known hotspots of PRDM9-directed histone methylation and DNA DSBs, thus proving their origin in the standard DSB repair pathway. A significant deficit of noncrossovers descending from asymmetric DSBs pointed to sister chromatids as an alternative template for their repair. The finding has implications for the molecular mechanism of hybrid sterility in mice.

scholarly journals Chromosome-wide characterization of meiotic noncrossovers (gene conversions) in mouse hybrids

Genetics ◽  
2020 ◽  
Vol 217 (1) ◽  
pp. 1-14
Author(s):  
Vaclav Gergelits ◽  
Emil Parvanov ◽  
Petr Simecek ◽  
Jiri Forejt
Keyword(s):  
Mus Musculus ◽  
Hybrid Sterility ◽  
Wide Distribution ◽  
Mus Musculus Domesticus ◽  
Dna Double Strand Breaks ◽  
Mus Musculus Musculus ◽  
Significant Deficit ◽  
Chromosome Substitution Strains ◽  
Gene Conversions

Abstract During meiosis, the recombination-initiating DNA double-strand breaks (DSBs) are repaired by crossovers or noncrossovers (gene conversions). While crossovers are easily detectable, noncrossover identification is hampered by the small size of their converted tracts and the necessity of sequence polymorphism. We report identification and characterization of a mouse chromosome-wide set of noncrossovers by next-generation sequencing of 10 mouse intersubspecific chromosome substitution strains. Based on 94 identified noncrossovers, we determined the mean length of a conversion tract to be 32 bp. The spatial chromosome-wide distribution of noncrossovers and crossovers significantly differed, although both sets overlapped the known hotspots of PRDM9-directed histone methylation and DNA DSBs, thus supporting their origin in the standard DSB repair pathway. A significant deficit of noncrossovers descending from asymmetric DSBs proved their proposed adverse effect on meiotic recombination and pointed to sister chromatids as an alternative template for their repair. The finding has implications for the molecular mechanism of hybrid sterility in mice from crosses between closely related Mus musculus musculus and Mus musculus domesticus subspecies.

scholarly journals Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2′-guanosine

2019 ◽  
Vol 47 (17) ◽  
pp. 9410-9422 ◽  
Author(s):  
Andrea M Kaminski ◽  
Kishore K Chiruvella ◽  
Dale A Ramsden ◽  
Thomas A Kunkel ◽  
Katarzyna Bebenek ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Steady State Kinetics ◽  
Ligase Iv ◽  
Template Dna ◽  
Dna Substrate

Abstract DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2′-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.

scholarly journals Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination

2002 ◽  
Vol 195 (3) ◽  
pp. 309-316 ◽  
Author(s):  
Robert E. Tillman ◽  
Andrea L. Wooley ◽  
Maureen M. Hughes ◽  
Tara D. Wehrly ◽  
Wojciech Swat ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Receptor Gene ◽  
Antigen Receptor ◽  
Double Strand Breaks ◽  
Recombination Reaction ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Recombination Signal Sequences ◽  
Dna Dsbs ◽  
Cleavage Step

Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRβ locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination.

scholarly journals An Oligomerized 53BP1 Tudor Domain Suffices for Recognition of DNA Double-Strand Breaks

2008 ◽  
Vol 29 (4) ◽  
pp. 1050-1058 ◽  
Author(s):  
Omar Zgheib ◽  
Kristopher Pataky ◽  
Juergen Brugger ◽  
Thanos D. Halazonetis
Keyword(s):  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Checkpoint Proteins ◽  
Strand Breaks ◽  
Histone H2ax ◽  
Dna Dsbs ◽  
Tudor Domain ◽  
Histone Core ◽  
Histone H2ax Phosphorylation ◽  
Oligomerization Domain

ABSTRACT 53BP1, the vertebrate ortholog of the budding yeast Rad9 and fission yeast Crb2/Rhp9 checkpoint proteins, is recruited rapidly to sites of DNA double-strand breaks (DSBs). A tandem tudor domain in human 53BP1 that recognizes methylated residues in the histone core is necessary, but not sufficient, for efficient recruitment. By analysis of deletion mutants, we identify here additional elements in 53BP1 that facilitate recognition of DNA DSBs. The first element corresponds to an independently folding oligomerization domain. Replacement of this domain with heterologous tetramerization domains preserves the ability of 53BP1 to recognize DNA DSBs. A second element is only about 15 amino acids long and appears to be a C-terminal extension of the tudor domain, rather than an independently functioning domain. Recruitment of 53BP1 to sites of DNA DSBs is facilitated by histone H2AX phosphorylation and ubiquitination. However, none of the 53BP1 domains/elements important for recruitment are known to bind phosphopeptides or ubiquitin, suggesting that histone phosphorylation and ubiquitination regulate 53BP1 recruitment to sites of DNA DSBs indirectly.

scholarly journals Modulation of Prdm9-controlled meiotic chromosome asynapsis overrides hybrid sterility in mice

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sona Gregorova ◽  
Vaclav Gergelits ◽  
Irena Chvatalova ◽  
Tanmoy Bhattacharyya ◽  
Barbora Valiskova ◽  
...  
Keyword(s):  
Meiotic Chromosome ◽  
Hybrid Sterility ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Recombination Hotspots ◽  
Chromosome Synapsis ◽  
Reproductive Isolation Mechanisms ◽  
Meiotic Recombination Hotspots ◽  
Isolation Mechanisms

Hybrid sterility is one of the reproductive isolation mechanisms leading to speciation. Prdm9, the only known vertebrate hybrid-sterility gene, causes failure of meiotic chromosome synapsis and infertility in male hybrids that are the offspring of two mouse subspecies. Within species, Prdm9 determines the sites of programmed DNA double-strand breaks (DSBs) and meiotic recombination hotspots. To investigate the relation between Prdm9-controlled meiotic arrest and asynapsis, we inserted random stretches of consubspecific homology on several autosomal pairs in sterile hybrids, and analyzed their ability to form synaptonemal complexes and to rescue male fertility. Twenty-seven or more megabases of consubspecific (belonging to the same subspecies) homology fully restored synapsis in a given autosomal pair, and we predicted that two or more DSBs within symmetric hotspots per chromosome are necessary for successful meiosis. We hypothesize that impaired recombination between evolutionarily diverged chromosomes could function as one of the mechanisms of hybrid sterility occurring in various sexually reproducing species.

Detecting, signalling and repairing DNA double-strand breaks

2001 ◽  
Vol 29 (6) ◽  
pp. 655-661 ◽  
Author(s):  
S. P. Jackson
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Double Strand Breaks ◽  
Model Organisms ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Site Specific ◽  
Strand Breaks ◽  
Dna Dsbs ◽  
Recombination Processes

DNA double-strand breaks (DSBs) can be generated by a variety of genotoxic agents, including ionizing radiation and radiomimetic chemicals. They can also occur when DNA replication complexes encounter other forms of DNA damage, and are produced as intermediates during certain site-specific recombination processes. It is crucial that cells recognize DSBs and bring about their efficient repair, because a single unrepaired cellular DSB can induce cell death, and defective DSB repair can lead to mutations or the loss of significant segments of chromosomal material. Eukaryotic cells have evolved a variety of systems to detect DNA DSBs, repair them, and signal their presence to the transcription, cell cycle and apoptotic machineries. In this review, I describe how work on mammalian cells and also on model organisms such as yeasts has revelaed that such systems are highly conserved throughout evolution, and has provided insights into the molecular mechanisms by which DNA DSBs are recognized, signalled and repaired. I also explain how defects in the proteins that function in these pathways are associated with a variety of human pathological states.

scholarly journals DNA-dependent macromolecular condensation drives self-assembly of the meiotic DNA break machinery

2020 ◽  
Author(s):  
Corentin Claeys Bouuaert ◽  
Stephen Pu ◽  
Juncheng Wang ◽  
Dinshaw J. Patel ◽  
Scott Keeney
Keyword(s):  
Self Assembly ◽  
Chromosome Structure ◽  
Coiled Coil ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Breakage ◽  
And Control

Formation of meiotic DNA double-strand breaks (DSBs) by Spo11 is tightly regulated and tied to chromosome structure, but the higher-order assemblies that execute and control DNA breakage are poorly understood. We address this question through molecular characterization of Saccharomyces cerevisiae RMM proteins (Rec114, Mei4 and Mer2)—essential, conserved components of the DSB machinery. Each subcomplex of Rec114–Mei4 (2:1 heterotrimer) or Mer2 (homotetrameric coiled coil) is monodisperse in solution, but they independently condense with DNA into dynamic, reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive condensation, which correlates with DSB formation in vivo. Condensates fuse into mixed Rec114–Mei4–Mer2 clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centers of DSB activity. We propose that multilayered control of Spo11 arises from recruitment of regulatory components and modulation of biophysical properties of the condensates.

scholarly journals The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks

2020 ◽  
Vol 2020 ◽  
pp. 1-12 ◽  
Author(s):  
Lei Zhao ◽  
Chengyu Bao ◽  
Yuxuan Shang ◽  
Xinye He ◽  
Chiyuan Ma ◽  
...  
Keyword(s):  
Dna Repair ◽  
Double Strand Breaks ◽  
Ionising Radiation ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Dsbs ◽  
Repair Pathways

Ionising radiation- (IR-) induced DNA double-strand breaks (DSBs) are considered to be the deleterious DNA lesions that pose a serious threat to genomic stability. The major DNA repair pathways, including classical nonhomologous end joining, homologous recombination, single-strand annealing, and alternative end joining, play critical roles in countering and eliciting IR-induced DSBs to ensure genome integrity. If the IR-induced DNA DSBs are not repaired correctly, the residual or incorrectly repaired DSBs can result in genomic instability that is associated with certain human diseases. Although many efforts have been made in investigating the major mechanisms of IR-induced DNA DSB repair, it is still unclear what determines the choices of IR-induced DNA DSB repair pathways. In this review, we discuss how the mechanisms of IR-induced DSB repair pathway choices can operate in irradiated cells. We first briefly describe the main mechanisms of the major DNA DSB repair pathways and the related key repair proteins. Based on our understanding of the characteristics of IR-induced DNA DSBs and the regulatory mechanisms of DSB repair pathways in irradiated cells and recent advances in this field, We then highlight the main factors and associated challenges to determine the IR-induced DSB repair pathway choices. We conclude that the type and distribution of IR-induced DSBs, chromatin state, DNA-end structure, and DNA-end resection are the main determinants of the choice of the IR-induced DNA DSB repair pathway.

scholarly journals Generation of Markerless Deletions in the Nosocomial PathogenClostridium difficileby Induction of DNA Double-Strand Breaks

2018 ◽  
Vol 85 (3) ◽  
Author(s):  
Elena-Stella Theophilou ◽  
Prerna Vohra ◽  
Maurice P. Gallagher ◽  
Ian R. Poxton ◽  
Garry W. Blakely
Keyword(s):  
Clostridium Difficile ◽  
Regulatory Elements ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Strand Breaks ◽  
Allelic Exchange ◽  
Genes Encoding

ABSTRACTClostridium difficileis an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterization of such clinically important bacteria is often hampered by lack of availability of suitable tools. Here, we describe the use of I-SceI to induce DNA double-strand breaks, which increase the frequency of allelic exchange and enable the generation of markerless deletions inC. difficile. The usefulness of the system is illustrated by the deletion of genes encoding putative AddAB homologues. The ΔaddABmutants are sensitive to ultraviolet light and the antibiotic metronidazole, indicating a role in homologous recombination and the repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of thefliCgene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyltransferase.IMPORTANCEMost sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution inClostridium difficile. Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions inC. difficileand expands the toolbox available for genetic analysis of this important anaerobic pathogen.

scholarly journals FOXO3a protects glioma cells against temozolomide-induced DNA double strand breaks via promotion of BNIP3-mediated mitophagy

2021 ◽  
Author(s):  
Chuan He ◽  
Shan Lu ◽  
Xuan-zhong Wang ◽  
Chong-cheng Wang ◽  
Lei Wang ◽  
...  
Keyword(s):  
Glioma Cells ◽  
Double Strand Breaks ◽  
C6 Glioma Cells ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Mitochondrial Superoxide ◽  
Ros Accumulation ◽  
Intracellular Ros ◽  
Dna Dsbs ◽  
U251 Cells

AbstractFOXO3a (forkhead box transcription factor 3a) is involved in regulating multiple biological processes in cancer cells. BNIP3 (Bcl-2/adenovirus E1B 19-kDa-interacting protein 3) is a receptor accounting for priming damaged mitochondria for autophagic removal. In this study we investigated the role of FOXO3a in regulating the sensitivity of glioma cells to temozolomide (TMZ) and its relationship with BNIP3-mediated mitophagy. We showed that TMZ dosage-dependently inhibited the viability of human U87, U251, T98G, LN18 and rat C6 glioma cells with IC50 values of 135.75, 128.26, 142.65, 155.73 and 111.60 μM, respectively. In U87 and U251 cells, TMZ (200 μM) induced DNA double strand breaks (DSBs) and nuclear translocation of apoptosis inducing factor (AIF), which was accompanied by BNIP3-mediated mitophagy and FOXO3a accumulation in nucleus. TMZ treatment induced intracellular ROS accumulation in U87 and U251 cells via enhancing mitochondrial superoxide, which not only contributed to DNA DSBs and exacerbated mitochondrial dysfunction, but also upregulated FOXO3a expression. Knockdown of FOXO3a aggravated TMZ-induced DNA DSBs and mitochondrial damage, as well as glioma cell death. TMZ treatment not only upregulated BNIP3 and activated autophagy, but also triggered mitophagy by prompting BNIP3 translocation to mitochondria and reinforcing BNIP3 interaction with LC3BII. Inhibition of mitophagy by knocking down BNIP3 with SiRNA or blocking autophagy with 3MA or bafilomycin A1 exacerbated mitochondrial superoxide and intracellular ROS accumulation. Moreover, FOXO3a knockdown inhibited TMZ-induced BNIP3 upregulation and autophagy activation. In addition, we showed that treatment with TMZ (100 mg·kg−1·d−1, ip) for 12 days in C6 cell xenograft mice markedly inhibited tumor growth accompanied by inducing FOXO3a upregulation, oxidative stress and BNIP3-mediated mitophagy in tumor tissues. These results demonstrate that FOXO3a attenuates temozolomide-induced DNA double strand breaks in human glioma cells via promoting BNIP3-mediated mitophagy.

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