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.
Highlights
DNA double strand breaks (DSBs), are among the most potent genotoxic lesions, being able to induce chromosomal rearrangements [1] and constituting a major challenge to genomic stability
From the analysis of the peptides associated with the RPA–DNA complex, beyond the RPA complex, three proteins were identified having significant Mascot scores in the Swiss-Prot/Uniprot KB database (Supplementary Figure 1a): X-ray repair cross-complementing protein 6 (XRCC6), known as KU70, a key player of nonhomologous end-joining (NHEJ), which rapidly binds DSBs [36]; heterogeneous nuclear ribonucleoprotein A 1, involved in various aspects of RNA metabolism and known to interact with telomeric DNA to regulate telomere length [37,38]; and HNRNPD, known as AUF1, a key factor in the regulation of mRNAs involved in proliferation, senescence and stress response, which consists of four different isoforms (p37, p40, p42, p45) deriving from alternative splicing [39]
The RBP HNRNPD was shown to regulate the mRNA fate of many DNA damage response (DDR) proteins in response to DNA damage [40]
Summary
DNA double strand breaks (DSBs), are among the most potent genotoxic lesions, being able to induce chromosomal rearrangements [1] and constituting a major challenge to genomic stability. To safeguard genome stability and increase survival, cells use two principal pathways for DSBs repair: nonhomologous end-joining (NHEJ) [5] and homologous recombination (HR) [6]. The main difference between these two pathways consists in the fact that NHEJ, by joining DNA ends irrespectively of their original sequence, is errorprone, whereas HR restores the correct information using the sister chromatid as a faithful template. MRE11, which is endowed of both endo and exonuclease activity, promotes the formation of minimally resected ends by nicking DNA in multiple positions flanking the breaks, acting in concert with the recently identified EXD2 exonuclease [10]. Following initial resection the EXOI nuclease and the DNA2 helicase, in complex with
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