Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

In vitro reconstitution of meiotic DNA double-strand break formation

ABSTRACT: In vitro reconstitution of meiotic DNA double-strand break formation

PROVIDER: PRJNA1184718 | ENA |

REPOSITORIES: ENA

ACCESS DATA

Json Xml

Similar Datasets

Reconstitution of SPO11-dependent double-strand break formation

Project description:Reconstitution of SPO11-dependent double-strand break formation

| PRJNA1150203 | ENA

Meiotic double-strand break mapping in spo11 mutants by S1Seq

Project description:Meiotic double-strand break mapping in spo11 mutants by S1Seq

| PRJNA631754 | ENA

In vitro reconstitution of meiotic DNA double-strand break formation

Project description:The Spo11 complex catalyzes the formation of DNA double-strand breaks (DSBs), initiating meiotic recombination-a process essential for fertility and genetic diversity. Although Spo11’s function has been known for 27 years, previous efforts to reconstitute DSB formation in vitro have been unsuccessful. Here, we biochemically characterize mouse SPO11 and TOP6BL protein complex and demonstrate that this complex cleaves DNA and covalently attaches to the 5' terminus of DNA breaks in vitro. Using a point-mutation strategy, we reveal that Mg2+ is essential for DNA cleavage activity of this complex in vitro, as confirmed by knock-in mice carrying a point mutation in SPO11 that disrupts its binding to Mg2+, thereby abolishing DSB formation. However, the activity of the SPO11 complex is ATP-independent. We also present evidence that the mouse SPO11 complex is biochemically distinct from the ancestral topoisomerase VI. Our findings establish a mechanistic framework for understanding the initial steps of meiotic recombination.

2025-01-20 | GSE281557 | GEO

Meiotic prophase length modulates Tel1-dependent DNA double-strand break interference

Project description:Meiotic prophase length modulates Tel1-dependent DNA double-strand break interference

| PRJNA1027830 | ENA

Meiotic double-strand break mapping in spo11 mutants by S1Seq

Project description:The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are non-randomly distributed across the genome. Here, we use S1Seq mapping to map the distribution of meiotic DSBs in spo11 mutant strains of Saccharomyces cerevisiae.

2020-05-12 | GSE150313 | GEO

ANKRD31 anchors meiotic double-strand break formation as a direct partner of REC114

Project description:ANKRD31 anchors meiotic double-strand break formation as a direct partner of REC114

| PRJNA487273 | ENA

Genomic features shaping the landscape of meiotic double-strand break hotspots in maize

Project description:Genomic features shaping the landscape of meiotic double-strand break hotspots in maize

| PRJNA328981 | ENA

Reconstitution of SPO11-dependent double-strand break formation

Project description:Homologous meiotic recombination starts with DNA double-strand breaks (DSBs) generated by SPO11 protein. SPO11 is critical for meiosis in most species but the DSBs it makes are also dangerous because of their mutagenic and gametocidal potential, so cells must foster beneficial functions of SPO11 while minimizing its risks. SPO11 mechanism and regulation remain poorly understood. Here we report reconstitution of DNA cleavage in vitro with purified recombinant mouse SPO11 bound to its essential partner TOP6BL. Similar to their yeast orthologs, SPO11–TOP6BL complexes are monomeric (1:1) in solution and bind tightly to DNA. Unlike in yeast, however, dimeric (2:2) assemblies of mouse SPO11–TOP6BL cleaves DNA to form covalent 5 prime attachments requiring SPO11 active site residues, divalent metal ions, and SPO11 dimerization. Surprisingly, SPO11 can also manifest topoisomerase activity by relaxing supercoils and resealing DNA that it has nicked. Structure modeling with AlphaFold3 illuminates the protein-DNA interface and suggests that DNA is bent prior to cleavage. Deep sequencing of in vitro cleavage products reveals a rotationally symmetric base composition bias that partially explains DSB site preferences in vivo. Cleavage is inefficient on complex DNA substrates, partly because SPO11 is readily trapped in DSB-incompetent (presumably monomeric) binding states that exchange slowly. However, cleavage is improved by using substrates that favor DSB-competent dimer assembly, or by fusing SPO11 to an artificial dimerization module. Our results inform a model in which intrinsically feeble dimerization restrains SPO11 activity in vivo, making it exquisitely dependent on accessory proteins that focus and control DSB formation so that it happens only at the right time and the right places.

2024-08-20 | GSE275291 | GEO

Genomic features shaping the landscape of meiotic double-strand break hotspots in maize [ChIP-Seq]

Project description:To generate the double-strand break (DSB) hotspot map in maize, we used a chromatin immunoprecipitation (ChIP) approach, in which chromatin from flowers containing zygotene meiocytes was enriched in fragments associated with RAD51.

2016-07-08 | GSE55701 | GEO

CXXC1 is not essential for normal DNA double-strand break formation and meiotic recombination in mouse

Project description:In most mammals, including mice and humans, meiotic recombination is determined by the meiosis specific histone methytransferase PRDM9, which binds to specific DNA sequences and trimethylates histone 3 at lysine-4 and lysine-36 at the adjacent nucleosomes. These actions ensure successful DNA double strand break formation and repair that occur on the proteinaceous structure forming the chromosome axis. The process of hotspot association with the axis after their activation by PRDM9 is poorly understood. Previously, we and others have identified CXXC1, an ortholog of S. cerevisiae Spp1 in mammals, as a PRDM9 interactor. In yeast, Spp1 is a histone methyl reader that links H3K4me3 sites with the recombination machinery, promoting DSB formation. Here, we investigated whether CXXC1 has a similar function in mouse meiosis. We created two Cxxc1 conditional knockout mouse to deplete CXXC1 generally in germ cells, and before the onset of meiosis. Surprisingly, male knockout mice were fertile, and the loss of CXXC1 in spermatocytes had no effect on hotspot trimethylation, double-strand break formation or repair. Our results demonstrate that CXXC1 is not an essential link between PRDM9-activated recombination hotspot sites and DSB machinery and that the hotspot recognition pathway in mouse is independent of CXXC1.

2018-08-29 | GSE116336 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data