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" endonukleasy * genetika   fyziologie   metabolismus   MeSH
• buněčné linie MeSH
• DNA vazebné proteiny genetika   MeSH
• DNA-topoisomerasy I metabolismus   MeSH
• G1 fáze * genetika   MeSH
• jednořetězcové zlomy DNA * MeSH
• lidé MeSH
• methylmethansulfonát toxicita   MeSH
• oprava DNA * MeSH
• peroxid vodíku farmakologie   toxicita   MeSH
• protein XRCC1 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• "flap" endonukleasy * MeSH
• DNA vazebné proteiny MeSH
• DNA-topoisomerasy I MeSH
• FEN1 protein, human MeSH Prohlížeč
• methylmethansulfonát MeSH
• peroxid vodíku MeSH
• protein XRCC1 MeSH
• XRCC1 protein, human MeSH Prohlížeč

Somatic hypermutation (SHM) and class switch recombination (CSR) diversify immunoglobulin (Ig) genes and are initiated by the activation-induced deaminase (AID), a single-stranded DNA cytidine deaminase thought to engage its substrate during RNA polymerase II (RNAPII) transcription. Through a genetic screen, we identified numerous potential factors involved in SHM, including elongation factor 1 homolog (ELOF1), a component of the RNAPII elongation complex that functions in transcription-coupled nucleotide excision repair (TC-NER) and transcription elongation. Loss of ELOF1 compromises SHM, CSR, and AID action in mammalian B cells and alters RNAPII transcription by reducing RNAPII pausing downstream of transcription start sites and levels of serine 5 but not serine 2 phosphorylated RNAPII throughout transcribed genes. ELOF1 must bind to RNAPII to be a proximity partner for AID and to function in SHM and CSR, and TC-NER is not required for SHM. We propose that ELOF1 helps create the appropriate stalled RNAPII substrate on which AID acts.

• Klíčová slova
• AID, ELOF1, RNA polymerase II, class switch recombination, somatic hypermutation, transcription,
• MeSH
• AICDA (aktivací indukovaná cytidindeamináza) MeSH
• B-lymfocyty * imunologie   metabolismus   MeSH
• cytidindeaminasa metabolismus   genetika   MeSH
• fosfoproteiny * genetika   metabolismus   MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• lidé MeSH
• myši knockoutované MeSH
• myši MeSH
• oprava DNA MeSH
• přesmyk imunoglobulinových tříd * MeSH
• RNA-polymerasa II metabolismus   genetika   MeSH
• somatická hypermutace imunoglobulinových genů * MeSH
• transkripční elongační faktory * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AICDA (aktivací indukovaná cytidindeamináza) MeSH
• cytidindeaminasa MeSH
• fosfoproteiny * MeSH
• RNA-polymerasa II MeSH
• transkripční elongační faktory * 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 genetika   MeSH
• genetická transkripce genetika   MeSH
• histony metabolismus   MeSH
• jednořetězcové zlomy DNA * MeSH
• lidé MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• oprava DNA genetika   MeSH
• oxidační stres genetika   MeSH
• peroxid vodíku toxicita   MeSH
• poly(ADP-ribosa)polymerasa 1 genetika   metabolismus   MeSH
• protein XRCC1 genetika   metabolismus   MeSH
• specifické proteázy ubikvitinu metabolismus   MeSH
• ubikvitinace fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• histony MeSH
• PARP1 protein, human MeSH Prohlížeč
• peroxid vodíku MeSH
• poly(ADP-ribosa)polymerasa 1 MeSH
• protein XRCC1 MeSH
• specifické proteázy ubikvitinu MeSH
• USP3 protein, human MeSH Prohlížeč
• XRCC1 protein, human MeSH Prohlížeč