Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1214218 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR32124500_1.fastq.gz | Fastqsanger.gz | |||
| SRR32124500_2.fastq.gz | Fastqsanger.gz | |||
| SRR32124501_1.fastq.gz | Fastqsanger.gz | |||
| SRR32124501_2.fastq.gz | Fastqsanger.gz | |||
| SRR32124502_1.fastq.gz | Fastqsanger.gz |
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Project description:Homology search is a central step of DNA double-strand break (DSB) repair by homologous recombination. How it operates in cells remains elusive. Here we developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from local to genome-wide search by increasing the probability of assembly of extensive NPFs. DSB end-tethering promotes coordinated homology search by opposite NPFs. Finally, an autonomous genetic element on chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals the mechanism of the progressive expansion of homology search orchestrated by chromatin organizers, long-range resection, end-tethering, specialized genetic elements, and that exploits the stiff NPF structure conferred by Rad51 oligomerization.
2024-07-29 | GSE272969 | GEO
Project description:Homologous recombination (HR) is an ubiquitous DNA double-strand break (DSB) repair mechanism. It entails a homology search step, carried out along a conserved RecA/Rad51-ssDNA nucleoprotein filament (NPF) assembled on each DSB ends. In contrast to the extensive knowledge of DNA damage checkpoint (DDC)-induced changes in chromatin composition and mobility, the questions of if, how, and to what extent a DSB impacts the spatial organization of chromatin, and whether this organization in turn influences the homology search process, remain ill-defined. Here we characterize two layers of spatial chromatin reorganization following DSB formation in S. cerevisiae. While cohesin folds chromosomes into cohesive arrays of ~20 kb-long chromatin loops as cells arrest in G2/M, the DSB-flanking regions interact locally in a resection- and 9-1-1 clamp-dependent manner, independently of cohesin, Mec1ATR, Rad52 and Rad51. This local structure blocks cohesin progression, constraining the DSB region at the base of a loop. Functionally, cohesin promotes DSB-dsDNA interactions and donor identification in cis, while inhibiting them in trans. This study identifies multiple direct and indirect ways by which cohesin regulates homology search during HR repair.
2021-07-21 | GSE179641 | GEO
Project description:Homologous recombination (HR) is an ubiquitous DNA double-strand break (DSB) repair mechanism. It entails a homology search step, carried out along a conserved RecA/Rad51-ssDNA nucleoprotein filament (NPF) assembled on each DSB ends. In contrast to the extensive knowledge of DNA damage checkpoint (DDC)-induced changes in chromatin composition and mobility, the questions of if, how, and to what extent a DSB impacts the spatial organization of chromatin, and whether this organization in turn influences the homology search process, remain ill-defined. Here we characterize two layers of spatial chromatin reorganization following DSB formation in S. cerevisiae. While cohesin folds chromosomes into cohesive arrays of ~20 kb-long chromatin loops as cells arrest in G2/M, the DSB-flanking regions interact locally in a resection- and 9-1-1 clamp-dependent manner, independently of cohesin, Mec1ATR, Rad52 and Rad51. This local structure blocks cohesin progression, constraining the DSB region at the base of a loop. Functionally, cohesin promotes DSB-dsDNA interactions and donor identification in cis, while inhibiting them in trans. This study identifies multiple direct and indirect ways by which cohesin regulates homology search during HR repair.
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Project description:DNA double-stranded breaks (DSBs) are an especially toxic form of damage that can be reversed without loss of genetic information by homology-directed repair (HDR), which copies from an intact template molecule. Finding a correct template within millions to billions of other DNA bases is termed homology search and is mediated by the protein RAD51. A major challenge in understanding homology search is the lack of tools to monitor search itself, rather than the end-stage outcome of HDR. We developed RAD51 proximity identification sequencing (RaPID-seq), a highly sensitive in cellulo method that marks all DNA searched by RAD51, regardless of whether it is chosen as the final template. We find that HDR in human cells is hierarchical. Only DSB-proximal sequences are searched, and even identical sequences outside the proximal window are not efficiently interrogated. Final recombination is unlocked by template homology but only at proximal sequences. We further find that RAD51 efficiently searches exogenously introduced DNA templates, such as those commonly used during genome editing, even those with no homology to the DSB site. Such highly abundant exogenous templates compete with search of endogenous genome templates. Our data reveal the invisible process of homology search and shed new light on fundamental mechanisms underlying genome editing.
2026-07-15 | GSE303129 | GEO
Project description:DNA double-stranded breaks (DSBs) are an especially toxic form of damage that can be reversed without loss of genetic information by homology-directed repair (HDR), which copies from an intact template molecule. Finding a correct template within millions to billions of other DNA bases is termed homology search and is mediated by the protein RAD51. A major challenge in understanding homology search is the lack of tools to monitor search itself, rather than the end-stage outcome of HDR. We developed RAD51 proximity identification sequencing (RaPID-seq), a highly sensitive in cellulo method that marks all DNA searched by RAD51, regardless of whether it is chosen as the final template. We find that HDR in human cells is hierarchical. Only DSB-proximal sequences are searched, and even identical sequences outside the proximal window are not efficiently interrogated. Final recombination is unlocked by template homology but only at proximal sequences. We further find that RAD51 efficiently searches exogenously introduced DNA templates, such as those commonly used during genome editing, even those with no homology to the DSB site. Such highly abundant exogenous templates compete with search of endogenous genome templates. Our data reveal the invisible process of homology search and shed new light on fundamental mechanisms underlying genome editing.
2026-07-15 | GSE303128 | GEO
Project description:DNA double-stranded breaks (DSBs) are an especially toxic form of damage that can be reversed without loss of genetic information by homology-directed repair (HDR), which copies from an intact template molecule. Finding a correct template within millions to billions of other DNA bases is termed homology search and is mediated by the protein RAD51. A major challenge in understanding homology search is the lack of tools to monitor search itself, rather than the end-stage outcome of HDR. We developed RAD51 proximity identification sequencing (RaPID-seq), a highly sensitive in cellulo method that marks all DNA searched by RAD51, regardless of whether it is chosen as the final template. We find that HDR in human cells is hierarchical. Only DSB-proximal sequences are searched, and even identical sequences outside the proximal window are not efficiently interrogated. Final recombination is unlocked by template homology but only at proximal sequences. We further find that RAD51 efficiently searches exogenously introduced DNA templates, such as those commonly used during genome editing, even those with no homology to the DSB site. Such highly abundant exogenous templates compete with search of endogenous genome templates. Our data reveal the invisible process of homology search and shed new light on fundamental mechanisms underlying genome editing.
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Project description:DNA double-stranded breaks (DSBs) are an especially toxic form of damage that can be reversed without loss of genetic information by homology-directed repair (HDR), which copies from an intact template molecule. Finding a correct template within millions to billions of other DNA bases is termed homology search and is mediated by the protein RAD51. A major challenge in understanding homology search is the lack of tools to monitor search itself, rather than the end-stage outcome of HDR. We developed RAD51 proximity identification sequencing (RaPID-seq), a highly sensitive in cellulo method that marks all DNA searched by RAD51, regardless of whether it is chosen as the final template. We find that HDR in human cells is hierarchical. Only DSB-proximal sequences are searched, and even identical sequences outside the proximal window are not efficiently interrogated. Final recombination is unlocked by template homology but only at proximal sequences. We further find that RAD51 efficiently searches exogenously introduced DNA templates, such as those commonly used during genome editing, even those with no homology to the DSB site. Such highly abundant exogenous templates compete with search of endogenous genome templates. Our data reveal the invisible process of homology search and shed new light on fundamental mechanisms underlying genome editing.
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Project description:Cohesin regulates homology search during recombinational DNA repair
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Project description:Homologous recombination (HR) is crucial for genetic exchange, accurate repair of DNA double-strand breaks and pivotal for genome integrity. HR uses homologous sequences for repair, but how homology search, the exploration of the genome for homologous DNA sequences, is conducted in the nucleus remains poorly understood. Here, we use time-resolved chromatin immunoprecipitations of repair proteins to monitor homology search in vivo. We found that homology search proceeds by a probing mechanism, which commences around the break and samples preferentially on the broken chromosome. However, elements thought to instruct chromosome loops mediate homology search shortcuts, and centromeres, which cluster within the nucleus, may facilitate homology search on other chromosomes. Our study thus revealed crucial parameters for homology search in vivo and emphasizes the importance of linear distance, chromosome architecture and proximity for recombination efficiency. 2 new custom ChIP-chip platforms used; both Nimblegen; differ in oligo density: (platform 1: 2006-07-18_Scerevisiae_ChIP_Stefan Jentsch MPI Biochemistry S.cerevisiae 385K Tiling Array Version 1) ( platform 2: 100304_Scer2_MS_Chip_Stefan Jentsch MPI Biochemistry S.cerevisiae 135K Tiling Array Version 2) ChIP-chip profiling of DSB repair factors (Rad51, Rad52, RPA, gamma-H2A) upon single inducible DSBs in S.cerevisiae
2013-03-21 | E-GEOD-44844 | biostudies-arrayexpress