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

Alfano, L
; 
Caporaso, A
; 
Altieri, A
; 
Dell'Aquila, M
; 
Landi, C
; 
Bini, L
; 
Pentimalli, F
; 
Giordano, A
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Genre
Journal Article
Date
2019-01-01
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Subject
Antineoplastic Agents
 Camptothecin
 Checkpoint Kinase 1
 Chromatin
 DNA
 DNA Breaks, Double-Stranded
 DNA End-Joining Repair
 DNA, Single-Stranded
 Genome, Human
 Genomic Instability
 HeLa Cells
 Heterogeneous-Nuclear Ribonucleoprotein D
 Heterogeneous-Nuclear Ribonucleoprotein U
 Histones
 Humans
 Nucleic Acid Conformation
 Nucleic Acid Hybridization
 Phosphorylation
 Phthalazines
 Piperazines
 RNA Polymerase II
 RNA, Small Interfering
 Recombinational DNA Repair
 Replication Protein A
 Ribonuclease H
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http://hdl.handle.net/20.500.12613/4356
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DOI
10.1093/nar/gkz076
Abstract
© The Author(s) 2019. 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 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.
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Oxford University Press (OUP)
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Nucleic Acids Research
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