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

Nucleic Acids Research
Luigi AlfanoAntonio Giordano

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 binds 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; AsiSI-induced DSB resection; and 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 S...Continue Reading

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Citations

Jul 4, 2020·Essays in Biochemistry·Ruth F Ketley, Monika Gullerova
Mar 7, 2020·International Journal of Molecular Sciences·Giulia Maria NavaFederico Lazzaro
Aug 6, 2019·Frontiers in Molecular Biosciences·Federica MariniAchille Pellicioli
Jan 18, 2020·Biology·Nicola AlessioUmberto Galderisi
Apr 11, 2021·Nature Cell Biology·Aline Marnef, Gaëlle Legube
May 25, 2021·Frontiers in Molecular Biosciences·Julie A KlaricStephanie Panier
May 29, 2021·DNA Repair·Byeong Jin YeSoo Youn Choi

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Datasets Mentioned

BETA
PXD012045

Methods Mentioned

BETA
PCR
pull down
pull-down
transfection
immunoprecipitation
ChIP
FACS
affinity purification
X-ray
flow cytometry

Key Resources (RRID) Mentioned

CVCL_0030
AB_2117338
AB_2616009
AB_185548
AB_331563

Software Mentioned

ImageJ
[UNK]
Mascot
CellQuest

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