1. MCM7 is ubiquitylated by RNF8 in vivo and in vitro
To detect protein ubiquitination in the ionizing radiation (IR) induced DNA damage response pathway, we applied stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry (MS) analysis in HeLa cells, which showed that the ubiquitination of MCM7 was obviously decreased, typically at Lys 145 (Supplementary Table 1). Due to E3 ligase RNF8 playing an essential role in DSB repair, we hypothesized that whether RNF8 can catalyze MCM7 ubiquitination. To test this hypothesis, H1299 cells were transiently transfected with plasmids expressing His-ubiquitin (Ub), myc-MCM7, and HA-RNF8. Western blotting analysis showed that polyubiquitin-conjugated MCM7 was bound to Ni-NTA resin (Figure 1A). Additionally, we observed the same result in HEK293T cells (Figure 1B). To confirm that RNF8 ubiquitylates MCM7, we performed a FLAG-M2 agarose immunoprecipitation assay. HEK293T cells were transiently transfected with plasmids expressing Flag-MCM7, HA-Ub, and RNF8, which showed that MCM7 was ubiquitinated by antibodies against Flag and Ub (Figure 1C). To further verify that RNF8 ubiquitylates MCM7 in vitro, we purified Flag-RNF8 from HEK293T cells as the E3 ligase, pET-Flag-MCM7 from the Escherichia coli strain Rosetta (Figure 1D) and Flag-MCM7 from HEK293T cells (Figure 1E) as the substrate. In vitro ubiquitination assay showed that RNF8 catalyzed the polyubiquitylation of MCM7 directly (Figure 1D-1E), but not the RING-inactive mutant RNF8 C403S (Figure1F). According to the HPLC-MS/MS results, the ubiquitination of MCM7 occurred mainly at Lys 28 and Lys 145. Next, we generated plasmids harboring site-specific mutations in MCM7, namely, MCM7-K28R, MCM7-K145R and MCM7-K28R+K145R. The results showed that RNF8 catalyzes the polyubiquitylation of MCM7 specifically on Lys 145 in vivo (Figure 1G) and in vitro (Figure 2A). Correspondingly, knockdown of RNF8 suppressed the polyubiquitylation of MCM7 in U2OS cells (Figure 2B). Together, we identified that the E3 ligase RNF8 contributes for the polyubiquitylation of MCM7. Interesting, we also detected that MCM7 is highly ubiquitinated in the absence of RNF8 overexpression, which may be catalyzed by endogenous RNF8 or other E3s.
2. RNF8 dependent polyubiquitylation of MCM7 occurs on chromatin in the late S phase of the cell cycle
In the late M and G1 phases, the MCM2-7 DNA helicase is loaded at chromatin origins and gradually dissociates from chromatin during DNA replication in the S phase, although the total amount of MCM7 protein in the nucleus remains relatively constant[32]. To identify the distribution of ubiquitinated MCM7, we performed a chromatin fractionation assay with CSK buffer. We found that polyubiquitylation of MCM7 catalyzed by RNF8 occurred mainly in chromatin (Figure 2C-2E). Given that the association of MCMs with chromatin is cell cycle regulated, we synchronized HeLa cells at the G1-S boundary with double thymidine block, which showed that cells entered S phase during 2-6 h and the G2-M phase during 8-10 h after block release respectively (Figure 2F). Then, we isolated chromatin extraction from synchronized HeLa cells by double thymidine block and performed M2 beads pull down analysis, which showed that polyubiquitylation of MCM7 increased greatly but transiently after 6 h of release (Figure 3A), while the total amount of chromatin-bound MCM7 gradually diminished from the G1 phase to late M phase of the cell cycle (Figure 3A). In synchronized HeLa cells blocked by HU, polyubiquitylation of MCM7 was transiently increased after 5 h of release (Figure 3B), suggesting that ubiquitylation of MCM7 also occurs in the late S phase. This is slightly different to thymidine double block, which showed polyubiquitylation of MCM7 was transiently increased after 6 h of release. This may be caused by the difference of cell cycle progression after block and release by thymidine and HU. In conclusion, we showed that polyubiquitylation of MCM7 mediated by RNF8 occurs only transiently in vivo and is mainly involved in the final stages of DNA replication. This is consistent with previous reports in S. cerevisiae and Xenopus[7,8]. Moreover, the polyubiquitylation of MCM7 also occurred at the G1-S boundary and in the late M phase, suggesting an unknown function in the cell cycle, which remains to be further investigated.
3. MCM7 is polyubiquitylated by RNF8 with K63-linked ubiquitin chains
Differently linked ubiquitin chains have distinct topologies and cellular functions. In S. cerevisiae (by SCFDia2) and X. laevis (by Cul2LRR1), MCM7 is modified with K48-linked ubiquitin chains, which leads to replisome disassembly in the late S phase[7,8]. MCM7 is catalyzed by TRAIP to form K6- and K63-linked ubiquitin chains, which promote the mitotic disassembly pathway in X. laevis[11]. To uncover the novel function of ubiquitinated MCM7 in human tumor cells, we detected the formation of the ubiquitin chains of MCM7 mediated by RNF8. To determine the half-life of MCM7, U2OS cells were treated with cycloheximide (CHX), an inhibitor of eukaryotic protein synthesis, while the total amount of MCM7 remained stable (Figure 3C). To determine whether proteasome activity influences the lifespan of MCM7, U2OS cells were treated with MG-132, while we did not detect obvious degradation (Figure 3D). OTU DUBs (ovarian tumor-associated proteases domain-containing proteins) recognize and hydrolyze specific ubiquitin chain types and can be used to identify the linkage types on a ubiquitinated substrate[33]. To investigate linkage-specific polyubiquitin conjugation of MCM7 catalyzed by RNF8, we performed OTU DUBs to cleave the ubiquitin chain of MCM7 in vitro. The results showed that OTUD1 greatly cleaved the polyubiquitylation chains of MCM7 (Figure 3E). OTUD1 is highly active and specifically cleaves the K63-linked ubiquitin chain[33], suggesting that MCM7 ubiquitination catalyzed by RNF8 forms the K63-linked ubiquitin chain. Consistent with this notion, the K63-linked ubiquitin chain of MCM7 was enriched by K63-UIM in U2OS cells (Figure 3F). In HEK293T cells, the overexpression of His-Ub mutant K63R plasmid decreased polyubiquitylation of MCM7mediated by RNF8 (Figure 3G). In conclusion, MCM7 is polyubiquitylated by RNF8 with K63-linked ubiquitin chains.
4. RNF168 and BRCAI promote the polyubiquitylation of MCM7
At double-strand breaks (DSBs) induced by IR, RNF8 is responsible for the initiation of K63-linked ubiquitylation in the DNA damage response, which is subsequently amplified by RNF168 for the further recruitment of BRCA1, which is the regulator of the DSB HR response[14-21]. During ICL repair-mediated unloading in Xenopus, BRCA1 acts upstream of MCM7 polyubiquitylation and recruits p97 to promote CMG unloading[34, 35]. Therefore, we determined whether RNF168 and BRCA1 promote the ubiquitylation of MCM7. We used HeLa cells for overexpression of plasmids containing myc-MCM7, His-Ub and Flag-RNF168 or Flag-BRCA1 and found that both promoted the ubiquitylation of MCM7 in vivo (Figure 4A and 4C). However, we did not detect RNF168 mediated polyubiquitylation in vitro (Figure 4B), suggesting the function of RNF168 may be not directly regulated. In chromatin fractionation, the results showed that polyubiquitylation of MCM7 mediated by RNF168 and BRCA1 occurred on chromatin (Figure 4D). In synchronized HeLa cells blocked by HU, polyubiquitylation of MCM7 promoted by RNF168 was at the G1/S boundary and slightly increase after 6-7h of block release (Figure 4E). In addition, polyubiquitylation of MCM7 catalyzed by BRCA1 was transiently increased after 7h of release (Figure 4F), suggesting that ubiquitylated MCM7 occurs mainly in the late S phase or at the S/G2 boundary. Together, these results show that RNF168 and BRCA1 play a role in promoting polyubiquitylation of MCM7 in chromatin during termination of DNA replication. Consistent with the result for RNF8, the polyubiquitylation of MCM7 mediated by RNF168 and BRCA1 also occurs at the G1-S boundary and in the late M phase. The molecular basis of this regulation remains to be determined.
5. Inhibition of replication by DNA damage obviously reduces polyubiquitylation of MCM7 mediated by RNF8
DNA damage caused by physical genotoxic agents and chemical agents can induce genome instability, which causes fork replication stalling or collapse, disturbs replication fork progression, and triggers cell cycle arrest. Aphidicolin is an inhibitor of DNA polymerase α that blocks DNA replication in the S phase[36]. IR can induce single-strand breaks (SSBs), DSBs, and base damage[37]. Doxorubicin (Adriamycin) HCl blocks DNA synthesis by inserting itself into DNA and inhibiting DNA topology isomerase II[38]. Actinomycin D inhibits the initiation of DNA replication in mammalian cells[39]. DNA topoisomerase II (Top2) modulates the topological state of double-stranded DNA and allows the completion of DNA replication[40]. Both etoposide and ICRF-193 inhibit the activity of Top2, blocking replication fork termination between replisomes and the accumulation of blocked forks on chromatin in the late S phase[41, 42].
To determine whether the polyubiquitylation of MCM7 occurs in the final stage of DNA synthesis, we treated HeLa cells with HU to synchronize the cell cycle. After 2.5 h of release, HeLa cells were treated with various physical genotoxic agents and chemical agents, causing early-S phase and late-S phase damage in DNA replication. Because CMG unloading involves polyubiquitylation of CMG’s MCM7 subunit, we detected the ubiquitinated MCM7 and CMG disassembly on chromatin after 4-7 h of block release. The results showed that DNA damage significantly reduces the polyubiquitylation of MCM7 but increases the amount of MCM2 and MCM7 in chromatin extraction (Figure 5A-5F), suggesting prolonged association of replicative CMG helicase with chromatin. At the same time, flow cytometric analysis also showed that DNA damage caused S phase blockade (Figure 6A). In particular, the Top2 inhibitor ICRF-193 arrested cells at the late S phase (Figure 6A), consistent with the decreased ubiquitination of MCM7 (Figure 5F), which further verified that the ubiquitination of MCM7 occurred at the end of DNA replication. These results suggest that DNA damage can significantly reduce the polyubiquitylation of MCM7 in late S phase, suggesting that polyubiquitylation of MCM7 occurs only when DNA replication can be completely duplicated.
6. Proteasome and Cdc48/p97 segregase are required for MCM7 deubiquitylation and CMG disassembly
In X. laevis and S. cerevisiae, MCM7 deubiquitylation during replication termination depends on p97/Cdc48/VCP segregase but not proteasomal degradation[7, 8]. MCM7 polyubiquitylation acts as the signal for p97-mediated extraction and unloading of the CMG complex from chromatin[7, 8,10,13]. To investigate the mechanism of MCM7 deubiquitylation in human tumor cells, we inhibited proteasomal degradation with MG-132 treatment, which showed increased ubiquitination of MCM7 and prolonged CMG association on chromatin in the late S phase (Figure 6B). Blocking p97-mediated polyubiquitylation segregation by NMS-873 also resulted in increased polyubiquitylation of MCM7 and prolonged association of the helicase components MCM2, CDC45 and PCNA with chromatin (Figure 6C). Moreover, flow cytometry analysis showed that cells treated with MG-132 or NMS-873 were arrested in the S phase (Figure 6D). Together, these results suggest that deubiquitylation of MCM7 mediated by p97 and proteasomal degradation could drive the disassembly of the replicative helicase during termination. Consistent with the results for S. cerevisiae, X. laevis, C. elegans embryos and mouse embryonic stem cells[7, 8,13], we identified that polyubiquitylation of MCM7 plays a conserved role in regulating replisome disassembly from chromatin in humans.
7. USP29 and ATXN3 promote MCM7 deubiquitylation and CMG disassembly
Substrate release from the p97 complex requires the cooperation of a DUB, which trims polyubiquitin to an oligoubiquitin chain that is then translocated through the pore[43]. To further explore the mechanism of CMG disassembly, we systematically screened the DUBs that are responsible for deubiquitylation of MCM7 in vitro and in vivo. We purified ubiquitinated MCM7 and 77 DUBs for the in vitro deubiquitylation assay, which showed that USP7, USP8, USP12, USP15, USP18, USP19, USP20, USP28, USP29, USP30, USP31, USP33, USP36, USP37, DUB3, USP45, OTUD1, ATXN3, JOSD2, and OTUD6A can deubiquitylate MCM7 (Figure S1A). Therefore, we designed shRNAs targeting these DUBs for knockdown experiments in vivo. Due to the lack of partial DUB plasmids, we also designed shRNAs targeting MINDY1, MINDY2, MINDY3, MINDY4, USP50, USP53 and ZRANB1. Then, HEK293T cells were used to package lentivirus, and U2OS cells were infected. The results showed that USP29, USP50, USP53, MINDY2 and ATXN3 could remove the polyubiquitylation of MCM7 in vivo (Figure S1B). To further identify the DUBs of MCM7, we knocked down USP29, USP50, USP53, ATXN3 and MINDY2 in U2OS cells with the indicated siRNAs and found that USP29 and ATXN3 were more efficient at catalyzing the deubiquitylation of MCM7 (Figure 7A). In U2OS cells synchronized by HU, knockdown of USP29 and ATXN3 increased the polyubiquitylation of MCM7 and prolonged its accumulation on chromatin. Moreover, we detected the inhibition of replisome disassembly (Figure 7B). Correspondingly, knocking down RNF8, USP29 and ATXN3 perturbed the cell cycle and arrested S phase progression (Figure 7C-7D). Therefore, USP29 and ATXN3 promote MCM7 deubiquitylation and CMG disassembly (Figure 7E).