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Strand-specific ChIP-seq at DNA breaks distinguishes single versus doubled stranded DNA binding and refutes single stranded nucleosomes

ABSTRACT: In a first step of DNA double-strand break (DSB) repair by homologous recombination, DNA ends are resected such that single-stranded DNA (ssDNA) overhangs are generated. ssDNA is specifically bound by RPA and other factors, which constitutes a ssDNA-compartment on damaged chromatin. The molecular organization of this ssDNA- as well as the adjacent dsDNA-compartment is crucial during DSB signaling and repair. However, data regarding the association of the most basic chromatin components – the nucleosomes – have been discrepant. Here, we use site-specific induction of DSBs and chromatin-immunoprecipitation followed by strand-specific sequencing to analyse in vivo binding of key DSB repair and signalling proteins to either the ssDNA- or dsDNA-compartment. In case of nucleosomes, we show that recently proposed ssDNA-nucleosomes are not a major, persistent species, but that nucleosomes eviction and DNA end resection are intrinsically coupled. These results support a model of separated dsDNA-nucleosome- and ssDNA-RPA-compartments during DSB signaling and repair.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE149807 | GEO | 2021/03/01

REPOSITORIES: GEO

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RNA processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

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Project description:Repair of DNA double-strand break (DSB) is critical for the maintenance of genome integrity. We have previously shown that a class of DSB-induced small RNAs (diRNAs) facilitates homologous recombination (HR)-mediated DSB repair in Arabidopsis thaliana. Here we show that INVOLVED IN DE NOVO 2 (IDN2), a double-stranded RNA (dsRNA) binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA-binding ARGONAUTE 2 (AGO2) leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from ssDNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair.

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RNA processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

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Role of 53BP1 in End Protection and DNA synthesis at DNA breaks [END-seq]

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Role of 53BP1 in End Protection and DNA synthesis at DNA breaks [ChIP-seq]

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