p97 Promotes a Conserved Mechanism of Helicase Unloading during DNA Cross-Link Repair

Abstract

Interstrand cross-links (ICLs) are extremely toxic DNA lesions that create an impassable roadblock to DNA replication. When a replication fork collides with an ICL, it triggers a damage response that promotes multiple DNA processing events required to excise the cross-link from chromatin and resolve the stalled replication fork. One of the first steps in this process involves displacement of the CMG replicative helicase (comprised of Cdc45, MCM2-7, and GINS), which obstructs the underlying cross-link. Here we report that the p97/Cdc48/VCP segregase plays a critical role in ICL repair by unloading the CMG complex from chromatin. Eviction of the stalled helicase involves K48-linked polyubiquitylation of MCM7, p97-mediated extraction of CMG, and a largely degradation-independent mechanism of MCM7 deubiquitylation. Our results show that ICL repair and replication termination both utilize a similar mechanism to displace the CMG complex from chromatin. However, unlike termination, repair-mediated helicase unloading involves the tumor suppressor protein BRCA1, which acts upstream of MCM7 ubiquitylation and p97 recruitment. Together, these findings indicate that p97 plays a conserved role in dismantling the CMG helicase complex during different cellular events, but that distinct regulatory signals ultimately control when and where unloading takes place.

FIG 1

p97 plays a critical role in ICL repair. (A) pICL schematic. Cross-linked nucleotides are shown in blue. Primer pairs are shown for the ICL locus (bp 25 to 132 from the ICL) and FAR locus (bp 2523 to 2622 from the ICL). (B) Model of ICL repair in Xenopus egg extracts. Parental DNA strands are shown in black, and nascent strands are shown in gray or red for emphasis. CMG, replicative helicase comprised of Cdc45, MCM2-7, and GINS. (C to F) pICL was replicated in extract supplemented with buffer (+Buffer), 100 μM NMS-873 (+NMS-873), or 100 μM NMS-873 and 50 μM ubiquitin (+NMS-873 +Ub). (C) DNA intermediates were digested with HincII or HincII and SapI and then resolved by native agarose gel electrophoresis and visualized by autoradiography. (D and E) Intermediates of replication and repair are indicated to the right. DNA synthesis (D) and ICL repair (E) were calculated from results shown in panel C. See Fig. S1 in the supplemental material for illustration of experimental replicates of ICL repair in NMS-873-treated reaction mixtures. (F) The presence of free ubiquitin in total extract was visualized by Western blotting with antiubiquitin antibodies.

FIG 2

p97 inhibition blocks CMG unloading. pICL was replicated in extract supplemented with buffer (+Buffer), 100 μM NMS-873 (+NMS-873), or 100 μM NMS-873 and 50 μM ubiquitin (+NMS-873 +Ub). (A) DNA intermediates were digested with AflIII and then resolved by denaturing PAGE and visualized by autoradiography. A schematic of the nascent leading-strand intermediates produced by the rightward-moving fork when it encounters the ICL is shown below (reprinted from reference with the publisher's permission). (B and C) ICL recruitment was analyzed by ChIP with the indicated antibodies. See Fig. S2 in the supplemental material for quantitation of nascent-strand products.

FIG 3

Polyubiquitylation of chromatin-bound MCM7. (A) Schematic of the plasmid pulldown assay. (B and C) pICL (B) or pControl (C) was allowed to replicate in extract supplemented with buffer (+Buffer), 100 μM NMS-873 (+NMS-873), or 75 μM MG-262 (+MG-262). At the indicated time points, plasmid DNA was isolated from extract by plasmid pulldown. DNA-bound proteins were then visualized by Western blotting with anti-MCM7 antibodies. (D) pICL was allowed to replicate in extract supplemented with buffer, NMS-873, MG-262, 50 μM ubiquitin (Ub), or 50 μM GST-tagged ubiquitin (GST-Ub), as indicated. Plasmid-bound proteins were isolated at 60 min and visualized as described for panels B and C. Blots shown are with 2% of total input (IN), “−DNA” control samples, and a 0-time point value corresponding to plasmid DNA in HSS prior to NPE addition.

FIG 4

GST-ubiquitin disrupts p97-mediated helicase unloading. (A and B) pICL was allowed to replicate in extract supplemented with buffer (+Buffer), 50 μM ubiquitin (Ub), or 50 μM GST-tagged ubiquitin (GST-Ub). Samples were analyzed by agarose gel electrophoresis to determine the efficiency of replication (A) and ICL repair (B) (13). (C) Nascent-strand products were analyzed by denaturing PAGE. (D and E) ICL recruitment was analyzed by ChIP with the indicated antibodies. See Fig. S6 in the supplemental material for quantitation of nascent-strand products.

FIG 5

Analysis of K48-linked ubiquitin chain removal. (A) Schematic of His₆-ubiquitin pulldown. (B) pICL was allowed to replicate in extract supplemented with His₆-ubiquitin (+His-Ub), His₆-ubiquitin containing no lysines (+His-Ub-NOK), His₆-ubiquitin containing an arginine substitution at lysine 48 (+His-Ub-K48R), or His₆-ubiquitin containing only lysine 48 (+His-Ub-K48O) immediately after initiating replication. Ubiquitylated proteins were isolated by His₆-ubiquitin pulldown at 20 min and then visualized by Western blotting with anti-MCM7 antibodies. (C and D) pICL (C) or pControl (D) was allowed to replicate in extract supplemented with His₆-ubiquitin and buffer (+Buffer), 100 μM NMS-873 (+NMS-873), or 75 μM MG-262 (+MG-262). At the indicated times, ubiquitylated proteins were isolated and visualized as described for panel B. Lanes 5, 9, and 13 in both panels are reproduced to the right for direct comparison of MCM7 ubiquitylation at 20 min. Note that Ni-NTA beads nonspecifically bound some unmodified MCM7, which was also present in “−His” control samples.

FIG 6

BRCA1 acts upstream of MCM7 ubiquitylation and p97 recruitment. (A and B) Samples from Fig. 1 (A) or Fig. 4 (B) were analyzed via BRCA1 ChIP. (C and D) pICL (C) or pControl (D) was allowed to replicate in mock- or BRCA1-depleted extract. DNA-bound proteins were then isolated by plasmid pulldown and visualized by Western blotting with anti-MCM7 antibodies. (E to G) Mock- and BRCA1-depleted samples from the same reaction mixture analyzed in panel C were also analyzed by ChIP with the indicated antibodies. (H) Autoubiquitylation of BRCA1 was tested with the indicated E2 ubiquitin-conjugating enzymes, or no E2 enzyme (–), and then visualized by Western blotting with antiubiquitin antibodies. (I) BRCA1 ubiquitylation reaction mixtures shown in panel H were also incubated with stalled CMG isolated from extract in a plasmid pulldown and then visualized with anti-MCM7 antibodies. Light and dark exposures of the same blot are shown.