Characterization of human Spartan/C1orf124, an ubiquitin-PCNA interacting regulator of DNA damage tolerance

Abstract

Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.

The DNA-binding box of human SPARTAN contributes to the targeting of Polη to DNA damage sites

“…Based on this observation, we concluded that the DNA-binding box is not essential for the targeting of SPARTAN. This also indicated that the folding of SPARTAN is not affected by this mutation since both the C-terminal UBZ and the PIP boxes are necessary for its correct targeting to PCNA foci [26]. Moreover, to exclude the possibility that the SPARTAN A mutant hetero-oligomerizes in vivo with its endogenous wild-type form, we repeated the experiment on a SPARTAN-depleted cell line as well ( Figure 3B).…”

“…Proteins were produced as described previously [26]. Proteins were eluted from the beads with 20 mM reduced glutathione in NT buffer, resulting in GST-Flag-Spartan proteins.…”

“…Cell competition-based survival assay was performed as described earlier [26] using GFPexpressing HEK293 cells as control. HEK293 and, in paralell, HEK293 Spartan shRNA-expressing cell lines were transfected with control or Flag-SPARTAN-expressing plasmids and mixed with GFPexpressing HEK293 cells as published previously [34].…”

“…We and others have described previously that Spartan has an important function at the stalled replication fork in the targeting of Polη to the site of DNA damage induced by UV irradiation [24][25][26][27][28]. Since it has also been suggested that Spartan is the human homologue of yeast WSS1, which is a DNAdependent protease [33], we decided to test whether SPARTAN can bind DNA and whether this biochemical activity affects its function after UV damage.…”

“…The targeting of SPARTAN to the stalled replication fork depends mainly on its PCNA-interacting (PIP) and ubiquitin-binding zinc finger (UBZ) domains, which can mediate its interaction with Ub-PCNA. Additionally, SPARTAN plays an important role in the correct targeting of Polη to the stalled replication fork; in its absence, UV-induced Polη foci formation is highly defective [24][25][26][27][28]. Other publications suggest that SPARTAN is responsible for the targeting of p97 (VCP, vasolinecontaining protein), which is an ATP-dependent segregase and facilitates the dissociation of Polη from the site of action [29,30].…”