Proteomics

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Depletion of the RNA binding protein HNRNPD impairs homologous recombination by inhibiting DNA-end resection and inducing R-loop accumulation


ABSTRACT: DNA double strand break (DSB) repair through homologous recombination (HR) is crucial to maintain genome stability. DSB resection generates a single strand DNA intermediate, which is crucial for the HR process. We used a synthetic DNA structure, mimicking a resection intermediate, as a bait to identify proteins involved in this process. Among these, LC/MS analysis identified the RNA binding protein, HNRNPD. We found that HNRNPD was able to bind chromatin, although this binding occurred independently of DNA damage. However, upon damage, HNRNPD re-localized to γH2Ax foci and its silencing impaired CHK1 S345 phosphorylation and the DNA end resection process. Indeed, HNRNPD silencing reduced the ssDNA fraction upon camptothecin treatment and AsiSI-induced DSB resection and reduced RPA32 S4/8 phosphorylation. CRISPR/Cas9-mediated HNRNPD knockout impaired in vitro DNA resection and sensitized cells to camptothecin and olaparib treatment. We found that HNRNPD interacts with the heterogeneous nuclear ribonucleoprotein SAF-A previously associated with DNA damage repair. HNRNPD depletion resulted in an increased amount of RNA:DNA hybrids upon DNA damage . Both the expression of RNase H1 and RNA pol II inhibition recovered the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells upon DNA damage, suggesting that RNA:DNA hybrid resolution likely rescues the defective DNA damage response of HNRNPD-depleted cells.

INSTRUMENT(S): LCQ Classic

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Claudia Landi  

LAB HEAD: Luca Bini

PROVIDER: PXD012045 | Pride | 2019-02-26

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
DNAdsRPAWT.1317.1317.2.dta Other
DNAdsRPAWT.1317.1317.2.pride.mgf.gz Mgf
DNAdsRPAWT.1581.1581.2.dta Other
DNAdsRPAWT.1581.1581.2.pride.mgf.gz Mgf
DNAdsRPAWT.RAW Raw
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Publications

Depletion of the RNA binding protein HNRNPD impairs homologous recombination by inhibiting DNA-end resection and inducing R-loop accumulation.

Alfano Luigi L   Caporaso Antonella A   Altieri Angela A   Dell'Aquila Milena M   Landi Claudia C   Bini Luca L   Pentimalli Francesca F   Giordano Antonio A  

Nucleic acids research 20190501 8


DNA double strand break (DSB) repair through homologous recombination (HR) is crucial to maintain genome stability. DSB resection generates a single strand DNA intermediate, which is crucial for the HR process. We used a synthetic DNA structure, mimicking a resection intermediate, as a bait to identify proteins involved in this process. Among these, LC/MS analysis identified the RNA binding protein, HNRNPD. We found that HNRNPD binds chromatin, although this binding occurred independently of DNA  ...[more]

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