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Genome-wide profiling of DNA repair proteins in single cells

ABSTRACT: Genome-wide profiling of DNA repair proteins in single cells

PROVIDER: PRJNA956510 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR24190400.fastq.gz	Fastqsanger.gz
	SRR24190401.fastq.gz	Fastqsanger.gz
	SRR24190402.fastq.gz	Fastqsanger.gz
	SRR24190403.fastq.gz	Fastqsanger.gz
	SRR24190404.fastq.gz	Fastqsanger.gz

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Genome-wide profiling of DNA repair proteins in single cells

Project description:Accurate repair of DNA damage is critical for maintenance of genomic integrity and cellular viability. Because damage occurs non-uniformly across the genome, single-cell resolution is required for proper interrogation, but sensitive detection has remained challenging. Here, we present a comprehensive analysis of repair protein localization in single cells using DamID and ChIC sequencing techniques. This study reports genome-wide binding profiles in response to DNA double-strand breaks induced by AsiSI, and explores variability in genomic damage locations and associated repair features in the context of spatial genome organization. By unbiasedly detecting repair factor localization, we find that repair proteins often occupy entire topologically associating domains, mimicking variability in chromatin loop anchoring. Moreover, we demonstrate the formation of multi-way chromatin hubs in response to DNA damage. Notably, larger hubs show increased coordination of repair protein binding, suggesting a preference for cooperative repair mechanisms. Together, our work offers new insights into the heterogeneous processes underlying genome stability in single cells.

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Project description:We recently developed a high-resolution genome wide assay for mapping DNA excision repair named eXcision Repair-sequencing (XR-seq) (GEO accession: GSE67941) We have now used this assay to assay the effect of chromatin state on DNA repair. Here we report the results of a time-course of the repair of the UV induced damages cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PPs] in normal human skin fibroblasts. Comparison of this data to histone modification and DNA-seq maps (ENCODE) revealed initial repair of both damages is enriched in open and active chromatin states, whereas repair in heterochromatic and repressed chromatin states is relatively low and persists to later time points. We performed XR-seq for two types of UV induced damages (CPD and (6-4)PP) at multiple time points after UV irradiation, in normal NHF1, and CS-B (CS1ANps3g2, GM16095) fibroblast cell lines. Two biological replicates were performed for each experiment in which independent independent cell populations were UV treated and subjected to XR-seq. For assaying CPD repair, cells were irradiated with 10J/m2 and for assaying (6-4)PP cells were irradiated with 20J/m2. Raw data for the 1h time points of (6-4)PP repair are the same as in GEO accession GSE67941).

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Genome-wide Transcription-coupled Repair in E.coli is Mediated by the Mfd Protein

Project description:Transcription-coupled DNA repair is widespread in nature having been found in organisms from E. coli to yeast to humans. In E. coli, the transcription-repair coupling factor (TRCF) encoded by the mfd gene recognizes RNA polymerase (RNAP) stalled at a damage site and recruits the excision repair proteins to the stalled RNAP and promotes damage recognition while displacing RNAP and the truncated transcript from the damage site and in doing so, enhances the rate of repair of the transcribed strand (TS). Recently, it has been reported that there are other E. coli proteins that mediate transcription-coupled repair (TCR) independently of the Mfd protein. To understand the relative contributions of various proteins to and mechanisms of TCR we used the recently developed nucleotide excision repair sequencing (XR-seq) method for genome-wide analysis of excision repair to generate excision repair maps of entire genomes of various E. coli strains with deletions of genes implicated in TCR. Our data show that the Mfd protein catalyzes TCR throughout the genome.

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