DVC1 (C1orf124) is a DNA damage–targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks

Abstract

Ubiquitin-mediated processes orchestrate critical DNA-damage signaling and repair pathways. We identify human DVC1 (C1orf124; Spartan) as a cell cycle-regulated anaphase-promoting complex (APC) substrate that accumulates at stalled replication forks. DVC1 recruitment to sites of replication stress requires its ubiquitin-binding UBZ domain and PCNA-binding PIP box motif but is independent of RAD18-mediated PCNA monoubiquitylation. Via a conserved SHP box, DVC1 recruits the ubiquitin-selective chaperone p97 to blocked replication forks, which may facilitate p97-dependent removal of translesion synthesis (TLS) DNA polymerase η (Pol η) from monoubiquitylated PCNA. DVC1 knockdown enhances UV light-induced mutagenesis, and depletion of human DVC1 or the Caenorhabditis elegans ortholog DVC-1 causes hypersensitivity to replication stress-inducing agents. Our findings establish DVC1 as a DNA damage-targeting p97 adaptor that protects cells from deleterious consequences of replication blocks and suggest an important role of p97 in ubiquitin-dependent regulation of TLS.

The ADP-ribosyltransferase PARP10/ARTD10 Interacts with Proliferating Cell Nuclear Antigen (PCNA) and Is Required for DNA Damage Tolerance

“…In conclusion, PARP10 seem to be part of a positive feedback loop that recognizes and amplifies the PCNA ubiquitination signal. This is strikingly similar to the situation previously described for Spartan/C1orf124, another reader of PCNA ubiquitination involved in translesion synthesis (52)(53)(54). It will be important to investigate if Spartan and PARP10 work together.…”

Control of p97 function by cofactor binding

“…In mammals, the motif is found in the SEP (Shp, eyesclosed, p47) domain-containing UBX proteins p47, p37, UBXD4 and UBXD5, where it is located N-terminally of the UBX domain [46], to which it is connected via a mainly unstructured linker. Further mammalian members are the UBX protein TUG (also known as ASPL) [77], UFD1 [50], DVC1 (also called Spartan, a homolog of yeast Wss1), a proteolytic enzyme that functions in DNA repair coupled to DNA replication [67,78], and the Derlin proteins DER1 and DER2 involved in endoplasmic reticulum associated protein degradation (ERAD) [66,[79][80][81]. Currently, only limited structural information on the SHP box-p97 interaction is available, and the exact binding site of the SHP box on the p97 N domain remains unclear.…”

TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

“…For EdU staining, cells were treated with 10-µM EdU for 30 min before fixation and then stained using the Click-iT Plus EdU Alexa Fluor 647 Imaging kit (Invitrogen) according to the manufacturer's instructions. Confocal microscopy and laser microirradiation was performed essentially as described previously (Mosbech et al, 2012). In brief, confocal images were acquired with a confocal microscope (LSM 780; Carl Zeiss) and mounted on a confocal laser-scanning microscope (Axiovert 100M; Carl Zeiss) equipped with a Plan Apochromat 40×/1.3 NA oil immersion objective using standard settings.…”

“…To generate cell lines stably expressing untagged TRA IP siR alleles, U2OS cells were transfected with TRA IP constructs cloned into either pLenti6-UBC-V5 (Life Technologies) or pLenti CMV/TO Hygro DEST, and positive clones were selected by incubation in medium containing Blasticidin S (Invitrogen) or Hygromycin (Life Technologies), respectively, for 14 d. A U2OS cell line stably expressing GFP-RPA1 and RFP-PCNA was described previously (Mejlvang et al, 2014). The GFP-polη cell line has been described previously (Mosbech et al, 2012). Unless otherwise indicated, the following drug concentrations were used: 15-µM MMC, 1-µM CPT, 30-µM cisplatin, and 2-mM HU.…”

“…To generate stable U2OS-GFP-TRA IP WT and *PIP (F466A), U2OS cells were cotransfected with GFP-TRA IP expression constructs together with pBabe-Puro and selected with puromycin, as described previously (Mosbech et al, 2012). To generate cell lines stably expressing untagged TRA IP siR alleles, U2OS cells were transfected with TRA IP constructs cloned into either pLenti6-UBC-V5 (Life Technologies) or pLenti CMV/TO Hygro DEST, and positive clones were selected by incubation in medium containing Blasticidin S (Invitrogen) or Hygromycin (Life Technologies), respectively, for 14 d. A U2OS cell line stably expressing GFP-RPA1 and RFP-PCNA was described previously (Mejlvang et al, 2014).…”