scholarly journals C. elegans DSB-3 Reveals Conservation and Divergence among Protein Complexes Promoting Meiotic Double-Strand Breaks

2021 ◽  
Author(s):  
Albert W Hinman ◽  
Hsin-Yi Yeh ◽  
Baptiste Roelens ◽  
Kei Yamaya ◽  
Alexander Woglar ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Protein Complexes ◽  
Meiotic Chromosome ◽  
Structured Illumination ◽  
Dna Breaks ◽  
Structured Illumination Microscopy ◽  
Strand Breaks ◽  
C Elegans ◽  
Double Strand Dna Breaks ◽  
Essential Components

Meiotic recombination plays dual roles in the evolution and stable inheritance of genomes: recombination promotes genetic diversity by reassorting variants, and it establishes temporary connections between pairs of homologous chromosomes that ensure for their future segregation. Meiotic recombination is initiated by generation of double-strand DNA breaks (DSBs) by the conserved topoisomerase-like protein Spo11. Despite strong conservation of Spo11 across eukaryotic kingdoms, auxiliary complexes that interact with Spo11 complexes to promote DSB formation are poorly conserved. Here, we identify DSB-3 as a DSB-promoting protein in the nematode Caenorhabditis elegans. Mutants lacking DSB-3 are proficient for homolog pairing and synapsis but fail to form meiotic crossovers. Lack of crossovers in dsb-3 mutants reflects a requirement for DSB-3 in meiotic DSB formation. DSB-3 concentrates in meiotic nuclei with timing similar to DSB-1 and DSB-2 (predicted homologs of yeast/mammalian Rec114/REC114), and DSB-1, DSB-2, and DSB-3 are interdependent for this localization. Bioinformatics analysis and interactions among the DSB proteins support the identity of DSB-3 as a homolog of MEI4 in conserved DSB-promoting complexes. This identification is reinforced by colocalization of pairwise combinations of DSB-1, DSB-2, and DSB-3 foci in structured illumination microscopy images of spread nuclei. However, unlike yeast Rec114, DSB-1 can interact directly with SPO-11, and in contrast to mouse REC114 and MEI4, DSB-1, DSB-2 and DSB-3 are not concentrated predominantly at meiotic chromosome axes. We speculate that variations in the meiotic program that have co-evolved with distinct reproductive strategies in diverse organisms may contribute to and/or enable diversification of essential components of the meiotic machinery.

scholarly journals Caenorhabditis elegans DSB-3 reveals conservation and divergence among protein complexes promoting meiotic double-strand breaks

2021 ◽  
Vol 118 (33) ◽  
pp. e2109306118
Author(s):  
Albert W. Hinman ◽  
Hsin-Yi Yeh ◽  
Baptiste Roelens ◽  
Kei Yamaya ◽  
Alexander Woglar ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Meiotic Recombination ◽  
Protein Complexes ◽  
Meiotic Chromosome ◽  
Structured Illumination ◽  
Dna Breaks ◽  
Structured Illumination Microscopy ◽  
Strand Breaks ◽  
Double Strand Dna Breaks ◽  
Essential Components

Meiotic recombination plays dual roles in the evolution and stable inheritance of genomes: Recombination promotes genetic diversity by reassorting variants, and it establishes temporary connections between pairs of homologous chromosomes that ensure their future segregation. Meiotic recombination is initiated by generation of double-strand DNA breaks (DSBs) by the conserved topoisomerase-like protein Spo11. Despite strong conservation of Spo11 across eukaryotic kingdoms, auxiliary complexes that interact with Spo11 complexes to promote DSB formation are poorly conserved. Here, we identify DSB-3 as a DSB-promoting protein in the nematode Caenorhabditis elegans. Mutants lacking DSB-3 are proficient for homolog pairing and synapsis but fail to form crossovers. Lack of crossovers in dsb-3 mutants reflects a requirement for DSB-3 in meiotic DSB formation. DSB-3 concentrates in meiotic nuclei with timing similar to DSB-1 and DSB-2 (predicted homologs of yeast/mammalian Rec114/REC114), and DSB-1, DSB-2, and DSB-3 are interdependent for this localization. Bioinformatics analysis and interactions among the DSB proteins support the identity of DSB-3 as a homolog of MEI4 in conserved DSB-promoting complexes. This identification is reinforced by colocalization of pairwise combinations of DSB-1, DSB-2, and DSB-3 foci in structured illumination microscopy images of spread nuclei. However, unlike yeast Rec114, DSB-1 can interact directly with SPO-11, and in contrast to mouse REC114 and MEI4, DSB-1, DSB-2, and DSB-3 are not concentrated predominantly at meiotic chromosome axes. We speculate that variations in the meiotic program that have coevolved with distinct reproductive strategies in diverse organisms may contribute to and/or enable diversification of essential components of the meiotic machinery.

scholarly journals Dynamic Architecture of DNA Repair Complexes and the Synaptonemal Complex at Sites of Meiotic Recombination

2017 ◽  
Author(s):  
Alexander Woglar ◽  
Anne M. Villeneuve
Keyword(s):  
Synaptonemal Complex ◽  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Strand Breaks ◽  
Dynamic Architecture ◽  
Blm Helicase ◽  
Differential Timing ◽  
Two Populations

SummaryMeiotic double-strand breaks (DSBs) are generated and repaired in a highly regulated manner to ensure formation of crossovers (COs) while also enabling efficient non-CO repair to restore genome integrity. Here we use Structured-Illumination Microscopy to investigate the dynamic architecture of DSB repair complexes at meiotic recombination sites in relationship to the synaptonemal complex (SC). DSBs resected at both ends are rapidly converted into inter-homolog repair intermediates harboring two populations of BLM helicase and RPA, flanking a single population of MutS γ. These intermediates accumulate until late pachytene, when repair proteins disappear from non-CO sites and CO-designated sites become enveloped by SC-central region proteins, acquire a second MutS γ population, and lose RPA. These and other data suggest that the SC protects CO intermediates from being dismantled inappropriately and promotes step-wise CO maturation by generating a transient CO-specific repair compartment, thereby enabling differential timing and outcome of repair at CO and non-CO sites

scholarly journals Factors that affect the location and frequency of meiosis-induced double-strand breaks in Saccharomyces cerevisiae.

Genetics ◽  
1995 ◽  
Vol 140 (1) ◽  
pp. 55-66 ◽  
Author(s):  
T C Wu ◽  
M Lichten
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Structure ◽  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Yeast Genome ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Double Strand Dna Breaks ◽  
A Site ◽  
Chromosomal Sequences

Abstract Double-strand DNA breaks (DSBs) initiate meiotic recombination in Saccharomyces cerevisiae. DSBs occur at sites that are hypersensitive in nuclease digests of chromatin, suggesting a role for chromatin structure in determining DSB location. We show here that the frequency of DSBs at a site is not determined simply by DNA sequence or by features of chromatin structure. An arg4-containing plasmid was inserted at several different locations in the yeast genome. Meiosis-induced DSBs occurred at similar sites in pBR322-derived portions of the construct at all insert loci, and the frequency of these breaks varied in a manner that mirrored the frequency of meiotic recombination in the arg4 portion of the insert. However, DSBs did not occur in the insert-borne arg4 gene at a site that is frequently broken at the normal ARG4 locus, even though the insert-borne arg4 gene and the normal ARG4 locus displayed similar DNase I hypersensitivity patterns. Deletions that removed active DSB sites from an insert at HIS4 restored breaks to the insert-borne arg4 gene and to a DSB site in flanking chromosomal sequences. We conclude that the frequency of DSB at a site can be affected by sequences several thousand nucleotides away and suggest that this is because of competition between DSB sites for locally limited factors.

scholarly journals Fine-Structure Mapping of Meiosis-Specific Double-Strand DNA Breaks at a Recombination Hotspot Associated With an Insertion of Telomeric Sequences Upstream of the HIS4 Locus in Yeast

Genetics ◽  
1996 ◽  
Vol 143 (3) ◽  
pp. 1115-1125 ◽  
Author(s):  
Fei Xu ◽  
Thomas D Petes
Keyword(s):  
Meiotic Recombination ◽  
Hydroxyl Groups ◽  
Sequence Specificity ◽  
Structure Mapping ◽  
Dna Breaks ◽  
Exonuclease Iii ◽  
Recombination Hotspot ◽  
Telomeric Sequences ◽  
Double Strand Dna Breaks ◽  
Double Strand Dna

Abstract Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand DNA breaks (DSBs). Using two approaches, we mapped the position of DSBs associated with a recombination hotspot created by insertion of telomeric sequences into the region upstream of HIS4. We found that the breaks have no obvious sequence specificity and localize to a region of ~50 bp adjacent to the telomeric insertion. By mapping the breaks and by studies of the exonuclease III sensitivity of the broken ends, we conclude that most of the broken DNA molecules have blunt ends with 3′-hydroxyl groups.

scholarly journals Competition Between Adjacent Meiotic Recombination Hotspots in the Yeast Saccharomyces cerevisiae

Genetics ◽  
1997 ◽  
Vol 145 (3) ◽  
pp. 661-670 ◽  
Author(s):  
Qing-Qing Fan ◽  
Fei Xu ◽  
Michael A White ◽  
Thomas D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Telomeric Sequence ◽  
Coding Region ◽  
Dna Breaks ◽  
Local Formation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Recombination Hotspots ◽  
Double Strand Dna Breaks ◽  
His4 Gene

In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes.

scholarly journals Double-strand DNA breaks recruit the centromeric histone CENP-A

2009 ◽  
Vol 106 (37) ◽  
pp. 15762-15767 ◽  
Author(s):  
Samantha G. Zeitlin ◽  
Norman M. Baker ◽  
Brian R. Chapados ◽  
Evi Soutoglou ◽  
Jean Y. J. Wang ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Double Strand Dna Breaks ◽  
Histone H3 Variant ◽  
Radiation Induced ◽  
Non Homologous End Joining

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair.

scholarly journals Numerical and Spatial Patterning of Yeast Meiotic DNA Breaks by Tel1

2016 ◽  
Author(s):  
Neeman Mohibullah ◽  
Scott Keeney
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Deep Sequencing ◽  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Spatial Patterning ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Context Dependent

AbstractThe Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are dangerous lesions that can disrupt genome integrity, so meiotic cells regulate their number, timing, and distribution. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to examine the contribution of the DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM) to this regulation in Saccharomyces cerevisiae. A tel1Δ mutant had globally increased amounts of Spo11-oligonucleotide complexes and altered Spo11-oligonucleotide lengths, consistent with conserved roles for Tel1 in control of DSB number and processing. A kinase-dead tell mutation also increased Spo11-oligonucleotide levels, but mutating known Tel1 phosphotargets on Hop1 and Rec114 did not. Deep sequencing of Spo11 oligonucleotides from tel1Δ mutants demonstrated that Tel1 shapes the nonrandom DSB distribution in ways that are distinct but partially overlapping with previously described contributions of the recombination regulator Zip3. Finally, we uncover a context-dependent role for Tel1 in hotspot competition, in which an artificial DSB hotspot inhibits nearby hotspots. Evidence for Tel1-dependent competition involving strong natural hotspots is also provided.

Combined expansion microscopy with structured illumination microscopy for analyzing protein complexes

2018 ◽  
Vol 13 (8) ◽  
pp. 1869-1895 ◽  
Author(s):  
Yongfu Wang ◽  
Zulin Yu ◽  
Cori K. Cahoon ◽  
Tari Parmely ◽  
Nancy Thomas ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Structured Illumination ◽  
Structured Illumination Microscopy

Timing of molecular events in meiosis in Saccharomyces cerevisiae: stable heteroduplex DNA is formed late in meiotic prophase

1993 ◽  
Vol 13 (1) ◽  
pp. 373-382 ◽  
Author(s):  
C Goyon ◽  
M Lichten
Keyword(s):  
Meiotic Prophase ◽  
Double Strand Breaks ◽  
Dna Breaks ◽  
Base Pairs ◽  
Heteroduplex Dna ◽  
Strand Breaks ◽  
Dna Structures ◽  
Double Strand Dna Breaks ◽  
Molecular Events ◽  
The Times

To better understand the means by which chromosomes pair and recombine during meiosis, we have determined the time of appearance of heteroduplex DNA relative to the times of appearance of double-strand DNA breaks and of mature recombined molecules. Site-specific double-strand breaks appeared early in meiosis and were formed and repaired with a timing consistent with a role for breaks as initiators of recombination. Heteroduplex-containing molecules appeared about 1 h after double-strand breaks and were followed shortly by crossover products and the first meiotic nuclear division. We conclude that parental chromosomes are stably joined in heteroduplex-containing structures late in meiotic prophase and that these structures are rapidly resolved to yield mature crossover products. If the chromosome pairing and synapsis observed earlier in meiotic prophase is mediated by formation of biparental DNA structures, these structures most likely either contain regions of non-Watson-Crick base pairs or contain regions of heteroduplex DNA that either are very short or dissociate during DNA purification. Two loci were examined in this study: the normal ARG4 locus, and an artificial locus consisting of an arg4-containing plasmid inserted at MAT. Remarkably, sequences in the ARG4 promoter that suffered double-strand cleavage at the normal ARG4 locus were not cut at significant levels when present at MAT::arg4. These results indicate that the formation of double-strand breaks during meiosis does not simply involve the specific recognition and cleavage of a short nucleotide sequence.

scholarly journals In vivo analysis of FANCD2 recruitment at meiotic DNA breaks in Caenorhabditis elegans

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Marcello Germoglio ◽  
Anna Valenti ◽  
Ines Gallo ◽  
Chiara Forenza ◽  
Pamela Santonicola ◽  
...  
Keyword(s):  
Dna Repair ◽  
Caenorhabditis Elegans ◽  
Genome Stability ◽  
Genetic Interaction ◽  
Model Systems ◽  
Dna Breaks ◽  
Strand Breaks ◽  
C Elegans ◽  
In Vivo Analysis

AbstractFanconi Anemia is a rare genetic disease associated with DNA repair defects, congenital abnormalities and infertility. Most of FA pathway is evolutionary conserved, allowing dissection and mechanistic studies in simpler model systems such as Caenorhabditis elegans. In the present study, we employed C. elegans to better understand the role of FA group D2 (FANCD2) protein in vivo, a key player in promoting genome stability. We report that localization of FCD-2/FANCD2 is dynamic during meiotic prophase I and requires its heterodimeric partner FNCI-1/FANCI. Strikingly, we found that FCD-2 recruitment depends on SPO-11-induced double-strand breaks (DSBs) but not RAD-51-mediated strand invasion. Furthermore, exposure to DNA damage-inducing agents boosts FCD-2 recruitment on the chromatin. Finally, analysis of genetic interaction between FCD-2 and BRC-1 (the C. elegans orthologue of mammalian BRCA1) supports a role for these proteins in different DSB repair pathways. Collectively, we showed a direct involvement of FCD-2 at DSBs and speculate on its function in driving meiotic DNA repair.

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