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 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.
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
MeSH terms
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Antineoplastic Agents / pharmacology
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Camptothecin / pharmacology
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Checkpoint Kinase 1 / genetics
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Checkpoint Kinase 1 / metabolism
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Chromatin / drug effects
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Chromatin / metabolism*
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Chromatin / ultrastructure
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DNA / drug effects
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DNA Breaks, Double-Stranded / drug effects
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DNA End-Joining Repair / drug effects
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DNA, Single-Stranded / genetics
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DNA, Single-Stranded / metabolism
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Genome, Human*
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Genomic Instability
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HeLa Cells
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Heterogeneous Nuclear Ribonucleoprotein D0
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Heterogeneous-Nuclear Ribonucleoprotein D / antagonists & inhibitors
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Heterogeneous-Nuclear Ribonucleoprotein D / genetics*
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Heterogeneous-Nuclear Ribonucleoprotein D / metabolism
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Heterogeneous-Nuclear Ribonucleoprotein U / genetics
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Heterogeneous-Nuclear Ribonucleoprotein U / metabolism
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Histones / genetics
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Histones / metabolism
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Humans
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Nucleic Acid Conformation
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Nucleic Acid Hybridization / drug effects
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Phosphorylation / drug effects
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Phthalazines / pharmacology
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Piperazines / pharmacology
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RNA Polymerase II / genetics
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RNA Polymerase II / metabolism
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RNA, Small Interfering / genetics
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RNA, Small Interfering / metabolism
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Recombinational DNA Repair* / drug effects
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Replication Protein A / genetics*
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Replication Protein A / metabolism
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Ribonuclease H / genetics
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Ribonuclease H / metabolism
Substances
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Antineoplastic Agents
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Chromatin
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DNA, Single-Stranded
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H2AX protein, human
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HNRNPD protein, human
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HNRNPU protein, human
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Heterogeneous Nuclear Ribonucleoprotein D0
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Heterogeneous-Nuclear Ribonucleoprotein D
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Heterogeneous-Nuclear Ribonucleoprotein U
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Histones
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Phthalazines
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Piperazines
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RNA, Small Interfering
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Replication Protein A
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DNA
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CHEK1 protein, human
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Checkpoint Kinase 1
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RNA Polymerase II
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RPA2 protein, human
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Ribonuclease H
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ribonuclease HI
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olaparib
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Camptothecin