Flap endonuclease 1 (FEN1)-dependent long-patch repair has been considered a minor sub-pathway of DNA single-strand break repair (SSBR), activated only when short-patch repair is not feasible. However, the significance of long-patch repair in living cells remains unclear. Here, we employed human RPE-1 cells with FEN1 deletion to compare the requirements for short- and long-patch pathways for the rapid repair of various types of DNA single-strand breaks (SSBs). We found that SSBs arising from abortive topoisomerase 1 activity are repaired efficiently without FEN1. In contrast, the rapid repair of SSBs arising during base excision repair following treatment with methyl methanesulphonate (MMS) or following treatment with hydrogen peroxide (H2O2) exhibits an unexpectedly high dependence on FEN1. Indeed, in G1 phase, FEN1 deletion slows the rate of SSBR to a similar or even greater extent than deletion of the short-patch repair proteins XRCC1 or POLβ. As expected, the combined deletion of FEN1 with XRCC1 or POLβ has an additive or synergistic effect, severely attenuating SSBR rates after MMS or H2O2 exposure. These data highlight an unanticipated requirement for FEN1 in the rapid repair of SSBs in human cells, challenging the prevailing view that long-patch repair is a minor sub-pathway of SSBR.

• MeSH
• Flap Endonucleases * genetics   physiology   metabolism   MeSH
• Cell Line MeSH
• DNA-Binding Proteins genetics   MeSH
• DNA Topoisomerases, Type I metabolism   MeSH
• G1 Phase * genetics   MeSH
• DNA Breaks, Single-Stranded * MeSH
• Humans MeSH
• Methyl Methanesulfonate toxicity   MeSH
• DNA Repair * MeSH
• Hydrogen Peroxide pharmacology   toxicity   MeSH
• X-ray Repair Cross Complementing Protein 1 MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Flap Endonucleases * MeSH
• DNA-Binding Proteins MeSH
• DNA Topoisomerases, Type I MeSH
• FEN1 protein, human MeSH Browser
• Methyl Methanesulfonate MeSH
• Hydrogen Peroxide MeSH
• X-ray Repair Cross Complementing Protein 1 MeSH
• XRCC1 protein, human MeSH Browser

RNA methylation, especially 6-methyladenosine (m6A)-modified RNAs, plays a specific role in DNA damage response (DDR). Here, we also observe that RNA modified at 8-methyladenosine (m8A) is recruited to UVA-damaged chromatin immediately after microirradiation. Interestingly, the level of m8A RNA at genomic lesions was reduced after inhibition of histone deacetylases and DNA methyltransferases. It appears in later phases of DNA damage response, accompanied by active DNA demethylation. Also, PARP inhibitor (PARPi), Olaparib, prevented adenosine methylation at microirradiated chromatin. PARPi abrogated not only m6A and m8A RNA positivity at genomic lesions, but also XRCC1, the factor of base excision repair (BER), did not recognize lesions in DNA. To this effect, Olaparib enhanced the genome-wide level of γH2AX. This histone modification interacted with m8A RNAs to a similar extent as m8A RNAs with DNA. Pronounced interaction properties we did not observe for m6A RNAs and DNA; however, m6A RNA interacted with XRCC1 with the highest efficiency, especially in microirradiated cells. Together, we show that the recruitment of m6A RNA and m8A RNA to DNA lesions is PARP dependent. We suggest that modified RNAs likely play a role in the BER mechanism accompanied by active DNA demethylation. In this process, γH2AX stabilizes m6A/m8A-positive RNA-DNA hybrid loops via its interaction with m8A RNAs. R-loops could represent basic three-stranded structures recognized by PARP-dependent non-canonical m6A/m8A-mediated DNA repair pathway.

• Keywords
• DNA demethylation, DNA repair, RNA methylation, base excision repair, epigenetics,
• MeSH
• Chromatin MeSH
• DNA Demethylation * MeSH
• DNA metabolism   MeSH
• DNA Methylation MeSH
• DNA Repair MeSH
• Poly(ADP-ribose) Polymerase Inhibitors * pharmacology   MeSH
• DNA Damage MeSH
• RNA genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• DNA MeSH
• Poly(ADP-ribose) Polymerase Inhibitors * MeSH
• RNA MeSH

Genetic defects in the repair of DNA single-strand breaks (SSBs) can result in neurological disease triggered by toxic activity of the single-strand-break sensor protein PARP1. However, the mechanism(s) by which this toxic PARP1 activity triggers cellular dysfunction are unclear. Here we show that human cells lacking XRCC1 fail to rapidly recover transcription following DNA base damage, a phenotype also observed in patient-derived fibroblasts with XRCC1 mutations and Xrcc1-/- mouse neurons. This defect is caused by excessive/aberrant PARP1 activity during DNA base excision repair, resulting from the loss of PARP1 regulation by XRCC1. We show that aberrant PARP1 activity suppresses transcriptional recovery during base excision repair by promoting excessive recruitment and activity of the ubiquitin protease USP3, which as a result reduces the level of monoubiquitinated histones important for normal transcriptional regulation. Importantly, inhibition and/or deletion of PARP1 or USP3 restores transcriptional recovery in XRCC1-/- cells, highlighting PARP1 and USP3 as possible therapeutic targets in neurological disease.

• MeSH
• DNA genetics   MeSH
• Transcription, Genetic genetics   MeSH
• Histones metabolism   MeSH
• DNA Breaks, Single-Stranded * MeSH
• Humans MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• DNA Repair genetics   MeSH
• Oxidative Stress genetics   MeSH
• Hydrogen Peroxide toxicity   MeSH
• Poly (ADP-Ribose) Polymerase-1 genetics   metabolism   MeSH
• X-ray Repair Cross Complementing Protein 1 genetics   metabolism   MeSH
• Ubiquitin-Specific Proteases metabolism   MeSH
• Ubiquitination physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Histones MeSH
• PARP1 protein, human MeSH Browser
• Hydrogen Peroxide MeSH
• Poly (ADP-Ribose) Polymerase-1 MeSH
• X-ray Repair Cross Complementing Protein 1 MeSH
• Ubiquitin-Specific Proteases MeSH
• USP3 protein, human MeSH Browser
• XRCC1 protein, human MeSH Browser