CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replication termination

Abstract

A key event during eukaryotic replication termination is the removal of the CMG helicase from chromatin. CMG unloading involves ubiquitylation of its Mcm7 subunit and the action of the p97 ATPase. Using a proteomic screen in Xenopus egg extracts, we identified factors that are enriched on chromatin when CMG unloading is blocked. This approach identified the E3 ubiquitin ligase CRL2^Lrr1, a specific p97 complex, other potential regulators of termination, and many replisome components. We show that Mcm7 ubiquitylation and CRL2^Lrr1 binding to chromatin are temporally linked and occur only during replication termination. In the absence of CRL2^Lrr1, Mcm7 is not ubiquitylated, CMG unloading is inhibited, and a large subcomplex of the vertebrate replisome that includes DNA Pol ε is retained on DNA. Our data identify CRL2^Lrr1 as a master regulator of replisome disassembly during vertebrate DNA replication termination.

Keywords: CMG; DNA replication; p97; replication termination; ubiquitin.

Figure 1.

Mcm7 ubiquitylation and p97 recruitment are termination-specific. (A) p[lacOx32] was incubated with LacR and then replicated until forks converged on the LacR array. At 18 min, IPTG and/or p97-i was added, as indicated. Chromatin-bound proteins were recovered by PPD at 18, 20, or 30 min and analyzed by Western blotting to detect Mcm7, Sld5, p97, Ufd1, and Orc2 (loading control). In parallel, reactions including radionucleotides were assembled to assess DNA replication (Supplemental Fig. S2K). (B) p[lacOx32] was incubated with LacR and then replicated in the presence or absence of p97-i and/or IPTG, as indicated. Chromatin-bound proteins were recovered by PPD at 30 min and blotted for Mcm7, Sld5, p97, Ufd1, and Orc2 (loading control). In parallel, reactions including radionucleotides were assembled to assess DNA replication (Supplemental Fig. S2L). The same protein samples were also analyzed in Figure 4B.

Figure 2.

CHROMASS analysis of proteins enriched on DNA in the presence of p97-i or Cul-i. (A) Sperm chromatin was replicated to completion in the presence of vehicle, p97-i, or Cul-i. Chromatin-bound proteins were recovered by chromatin spin-downs and analyzed by MS. For each protein detected in the screen, fold enrichment relative to the vehicle control was calculated for p97-i or Cul-i treatment. Significant hits are indicated with colored markers and grouped according to whether they were a hit in both conditions (group I), only in the presence of p97-i (group II), or only in the presence of Cul-i (group III). In parallel, a set of control experiments was performed in which replication was blocked by Geminin treatment (Supplemental Fig. S3C,D). (B) Heat map showing the mean of the z-scored log₂ LFQ intensity from four independent replicates described in A. LFQ values are arbitrary units, which reflect the abundance of different proteins recovered from chromatin in each condition. All proteins that were significantly up-regulated with p97-i and/or Cul-i (84 in total) (see the Materials and Methods) were subjected to nonsupervised hierarchical clustering. The main clusters corresponding to group I (enhanced binding with p97-i or Cul-i), group II (enhanced binding with p97-i only), or group III (enhanced binding with Cul-i only) were extracted and reclustered. (C) Mean log₂ LFQ intensity across all four replicates is shown for Cdc45, GINS (Psf1, Psf2, Psf3, and Sld5), and Mcm2–7 across all conditions assessed by MS, including Geminin treatment. Error bars represent standard deviation. (D) As in C but for Mcm10, Claspin, Timeless–Tipin (Tim and Tipin), And-1, DNA Pol ε (Dpoe1, Dpoe2, and Dpoe4), Ctf18–Ctf8–Dcc1, Dna2 (Dna2l), and DNA Pol α/primase complex (Dpola, Dpola2, Pri1, and Pri2). (E) As in C but for components of potential Cullin ligases, the ligase CRL2^Lrr1 (Cul2, Lrr1, EloB, and EloC), and Cul4a and Cul4b scaffolds and interacting proteins (Cul4a, Cul4b, Dda1, and Ddb1) as well as putative DCAFs (Dcaf17 and Brwd3) that are expected to form CRL4^Dcaf17 and CRL4^Brwd3. (F) As in C but for p97 and known substrate adapters: Ufd1–Npl4, Ubxn7, and Aspc1.

Figure 3.

Depletion of Lrr1 eliminates Mcm7 polyubiquitylation and inhibits CMG unloading. (A) Mock-depleted (mock) and Lrr1-depleted (ΔLrr1) extracts were blotted with Cul2 and Lrr1 antibodies. The bands corresponding to Cul2 and Lrr1 are marked with arrowheads. Over 90% of Lrr1 was depleted. (B) The extracts from A were used to replicate plasmids in the presence or absence of p97-i and/or Cul-i. Radionucleotides were included, and the relative extent of replication and decatenation was quantified at different time points (as in Supplemental Fig. S2H–J). (C) In parallel to B, reactions lacking radionucleotides were assembled. At 30 min, chromatin-bound proteins were recovered by PPD and blotted for Mcm7, Cdc45, Cul2, Lrr1, EloC, p97, Ufd1, and Orc2 (loading control). The same samples were also used in Supplemental Figure S7F. (D) Immunodepleted extracts were analyzed as in A except that EloC was also analyzed. Over 90% of Lrr1 was depleted. (E) p[lacOx32] was incubated with LacR and then replicated in Lrr1-depleted (ΔLrr1) extracts from D until forks converged on the LacR array. At 17 min, mock- or Lrr1-depleted extract was added along with IPTG to induce termination in the presence or absence of p97-i. Chromatin-bound proteins were recovered by PPD at time points between 17 and 40 min and then analyzed by Western blotting using antibodies against Mcm7, Mcm6, Cdc45, Sld5, Orc2, and Histone H3 (H3). Orc2 served as a loading control for the Mcm7 blot, and Histone H3 served as the loading control for the other blots. (F) Quantification of Mcm7, Mcm6, Cdc45, Sld5, Orc2, and Histone H3 protein recovery from E (see the Materials and Methods). As expected, in the presence p97-i, the unmodified form of Mcm7 disappeared in the presence of mock-depleted extract due to ubiquitylation (green graph) but not in LRR1-depleted extract, where ubiquitylation is inhibited (blue graphs).

Figure 4.

CRL2^Lrr1 is specifically recruited during termination. (A) p[lacOx32] was incubated with LacR and then replicated until forks converged on the LacR array. At 18 min, IPTG was added to induce termination or withheld to maintain the block along with either p97-i or Cul-i to block CMG unloading or vehicle control. Chromatin-bound proteins were recovered by PPD at 18 or 28 min and analyzed by Western blotting to detect Cul2, Lrr1, and Orc2 (loading control). In parallel, reactions including radionucleotides were assembled and analyzed to assess replication (Supplemental Fig. S7A). Samples are the same as those used in Figure 5, A and B. (B) The same samples described in Figure 1B were blotted for Cul2, Lrr1, and Orc2 (loading control). (C) Termination was induced and monitored as in A except that, after IPTG addition, chromatin was recovered by PPD at 17 or 18.5 min and every 30 sec thereafter and blotted for Cul2, Mcm7, Cdc45, Mcm6, and Histone H3 (H3; loading control). (D) Quantification of Cul2 binding, Mcm6 and Cdc45 dissociation, and Mcm7 ubiquitylation/dissociation from the Western blots shown in C and two additional experimental replicates (see the Materials and Methods). The mean is plotted, and error bars represent the standard deviation. Only samples lacking p97-i (even-numbered lanes in C) are quantified. (E) Plasmid DNA was incubated in egg extracts with p97-i and either vehicle or Cdc7-i. Chromatin-bound proteins were recovered by PPD after 30 min and blotted for Cul2, Lrr1, EloC, Mcm7, Mcm6, Sld5, Cdc45, and Orc2 (loading control).

Figure 5.

CRL2^Lrr1 is involved in disassembly of a vertebrate RPC. (A) The samples in Figure 4A were blotted for Mcm7, Cdc45, Sld5, Claspin, Mcm10, DNA Pol ε, Ctf18, Dna2, Traip, Timeless, Tipin, and Orc2 (loading control). In parallel, reactions including radionucleotides were assembled and analyzed to assess replication (Supplemental Fig. S7A). Traip was not detected in the input, but the band shown was depleted from extracts by immunoprecipitation using the Traip antibody (data not shown). (B) The same samples analyzed in A were also analyzed by Western blotting to detect DNA Pol α, And-1, and PCNA. (C) Plasmids were replicated in Lrr1-depleted extracts. At the indicated times, chromatin-bound proteins were recovered by PPD and blotted for Mcm7, Mcm6, Cdc45, Claspin, Timeless, Pol ε, Ctf18, Orc2, and Histone H3 (H3). Orc2 served as a loading control for Mcm7, Mcm6, and Cdc45, whereas Histone H3 served as a loading control for Claspin, Timeless, Pol ε, and Ctf18.

Figure 6.

Model for unloading of a vertebrate RPC by CRL2^Lrr1. (A) During replication, replication forks are bound by a vertebrate RPC (vRPC) that includes CMG, Mcm10, DNA Pol ε, Timeless/Tipin (Tim/Tipin), Claspin, Ctf18–RLC (not shown), Dna2 (not shown), Traip (not shown), and PCNA. (B) vRPCs pass each other and remain associated with CMGs as they translocate along ssDNA. (C) vRPCs translocate over the downstream lagging strands and encircle dsDNA. This prevents CMG and DNA Pol ε from blocking Okazaki fragment processing and allows binding by DNA Pol δ, which performs strand displacement synthesis. The presence of vRPCs on dsDNA leads to binding of And-1 and CRL2^Lrr1, which ubiquitylates Mcm7. If the replisome encounters a long downstream 5′ flap at the penultimate Okazaki fragment prior to unloading, reinitiation and unwinding by CMG and DNA synthesis by DNA Pol ε may be triggered. (D) vRPCs are extracted from chromatin by p97.