Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replication termination James M. Dewar,1,4,6 Emily Low,1,6 Matthias Mann,2 Markus Räschle,2,5 and Johannes C. Walter1,3 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA; 2Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; 3Howard Hughes Medical Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA 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 CRL2Lrr1, a specific p97 complex, other potential regulators of termi- nation, and many replisome components. We show that Mcm7 ubiquitylation and CRL2Lrr1 binding to chromatin are temporally linked and occur only during replication termination. In the absence of CRL2Lrr1, 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 CRL2Lrr1 as a master regulator of replisome disassembly during verte- brate DNA replication termination. [Keywords: DNA replication; replication termination; p97; CMG; ubiquitin] Supplemental material is available for this article. Received October 9, 2016; revised version accepted January 30, 2017. In eukaryotic cells, genomic DNA replication can be loaded onto DNA by the five-subunit RFC complex. divided into four phases: licensing, initiation, elongation, When the 3′ end of one Okazaki fragment encounters and termination (Siddiqui et al. 2013; Bell and Labib 2016). the 5′ end of another, DNA Pol δ displaces the RNA– During licensing, which occurs in the G1 phase of the cell DNA primer, and the resulting 5′ flap is removed by cycle, ORC, Cdc6, and Cdt1 load pairs of Mcm2–7 ATPas- FEN1 followed by ligation by DNA ligase I (Burgers es at each origin of replication. In vertebrates, ∼50,000 or- 2009). Long flaps with secondary structure are removed igins are licensed genome-wide (Prioleau and MacAlpine by the helicase/nuclease Dna2. During termination, 2016). Initiation occurs at the G1/S transition when replisomes converge, DNA synthesis is completed, the Cdc45 and the GINS complex (consisting of Sld5, Psf1, replisome is disassembled, and daughter duplexes are dec- Psf2, and Psf3) associate with Mcm2–7 to form active atenated. Importantly, a single defective termination CMG helicases. CMG encircles and travels along the lead- event has the potential to disrupt chromosome segrega- ing strand template (Fu et al. 2011; Kang et al. 2012). Dur- tion and cause genome instability, whereas failure to ini- ing elongation, the leading strand is copied by DNA tiate DNA synthesis at one or a few origins is readily polymerase ε (Pol ε). The lagging strand is synthesized as mitigated by firing of other origins. Despite the impor- ∼150-nucleotide Okazaki fragments (Burgers 2009). Each tance of replication termination, this process has received Okazaki fragment is initiated by the DNA Pol α/primase little attention compared with other stages of the replica- complex, which makes a short RNA–DNA primer that tion cycle. is extended by DNA Pol δ. DNA Pol δ acquires processiv- Recently, considerable progress has been made in un- ity by interacting with the sliding clamp PCNA, which is derstanding the events underlying replication termina- tion in eukaryotic cells. Use of a vertebrate cell-free system that supports site-specific and synchronous Present addresses: 4Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA; 5Department of Molecular Genetics, Techni- cal University of Kaiserslautern, 67653 Kaiserslautern, Germany. © 2017 Dewar et al. This article is distributed exclusively by Cold Spring 6These authors contributed equally to this work. Harbor Laboratory Press for the first six months after the full-issue publi- Corresponding authors: [email protected], markus. cation date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After [email protected] six months, it is available under a Creative Commons License (At- Article published online ahead of print. Article and publication date are tribution-NonCommercial 4.0 International), as described at http://creati- online at http://www.genesdev.org/cgi/doi/10.1101/gad.291799.116. vecommons.org/licenses/by-nc/4.0/. GENES & DEVELOPMENT 31:275–290 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/17; www.genesdev.org 275 Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Dewar et al. termination revealed that the leading strands of converg- p97 recruitment occur only when forks terminate. Our ing replication forks pass each other without stalling data suggest that convergence of CMGs during replication and that they are rapidly ligated to downstream Okazaki termination triggers de novo CRL2Lrr1 recruitment, fragments (Dewar et al. 2015). These observations are in Mcm7 ubiquitylation, and coordinated unloading of a contrast to termination of SV40 DNA tumor virus replica- large replisome subcomplex by p97. tion, where forks stall (Levine et al. 1970; Tapper and DePamphilis 1978; Seidman and Salzman 1979). More- over, converging CMG helicases are not unloaded until af- Results ter the leading strand of one fork has been ligated to the Mcm7 ubiquitylation and p97 chromatin recruitment downstream Okazaki fragment of the converging fork, im- are termination-specific events plying that CMG is unloaded from dsDNA (Dewar et al. 2015). Most likely, converging CMGs pass each other To study the mechanism of CMG unloading during repli- and continue translocating until they reach a downstream cation termination, we replicated a plasmid in Xenopus Okazaki fragment. They then appear to pass over the egg extracts and analyzed binding of replisome proteins ssDNA–dsDNA junction and probably translocate along by plasmid pull-down (PPD). To this end, magnetic beads dsDNA, as shown previously for purified Mcm2–7 and were coated with lac repressor (LacR) and incubated CMG complexes (Kaplan et al. 2003; Kang et al. 2012), be- with the chromatinized plasmid DNA in ice-cold buffer. fore being unloaded. The LacR-coated beads efficiently bound DNA in a se- Important aspects of CMG unloading have been re- quence-independent manner (Budzowska et al. 2015), vealed. Late in S phase, the Mcm7 subunit of CMG under- which allowed us to isolate the plasmid and examine goes K48-linked polyubiquitylation by a Cullin RING E3 chromatin-bound proteins by Western blotting (Supple- ubiquitin ligase (Maric et al. 2014; Moreno et al. 2014). mental Fig. S1A). As shown previously for sperm chroma- The ligase corresponds to SCFDia2 in yeast but remains un- tin replicating in egg extracts (Moreno et al. 2014), CMG known in higher eukaryotes. In the absence of SCFDia2, unloading from plasmid was blocked by the inhibitor CMG is retained on chromatin along with Ctf4/And-1 MLN4924 (“Cul-i”)(Supplemental Fig. S1A, cf. lanes 2 (also known as Wdhd1) and Csm3/Timeless (vertebrate and 4), which inhibits the activity of Cullin E3 ubiquitin names after slash), which stably interact with CMG as ligases by preventing their neddylation (Supplemental part of a “replisome progression complex” (RPC) that Fig. S1B, cf. lanes 2 and 3, Cul2 blot). CMG unloading also includes Tof1/Tipin, Pol α, Mcm10, Mrc1/Claspin, was also blocked by the p97 inhibitor NMS-873 FACT, and Topoisomerase I. These observations suggest (“p97-i”)(Supplemental Fig. S1A, lane 3). In contrast, bor- that Mcm7 ubiquitylation is the trigger for unloading tezomib had no effect (Supplemental Fig. S1A, lane 5), in- and disassembly of CMG and other RPC proteins. Yeast dicating that proteasome activity is not required. In the mutants lacking Dia2 (dia2Δ) show signs of genomic in- presence of p97-i but not Cul-i, Mcm7 appeared as a series stability, suggesting that CMG unloading is essential to of high-molecular-weight species (Supplemental Fig. S1A, preserve genomic stability (Blake et al. 2006; Koepp lanes 3,4), which represent polyubiquitin chains (Supple- et al. 2006). CMG unloading also depends on the p97 mental Fig. S1B) that were primarily K48-linked (Supple- ATPase (Franz et al. 2011; Maric et al. 2014; Moreno mental Fig. S1C). The polyubiquitin chains observed in et al. 2014), a “segregase” that extracts polyubiquitylated the presence of p97-i were abolished upon coaddition of proteins from their local environment in collaboration Cul-i, indicating that they represent Cullin-dependent with different adaptor proteins (Meyer et al. 2012; Buch- ubiquitylation (Supplemental Fig. S1A, lane 10). Impor- berger et al. 2015). Unloading may also require Ufd1 and tantly, when CMG unloading was blocked by p97-i, fork Npl4 (Franz et al. 2011), which form a heterodimer that convergence, the completion of DNA synthesis, and dec- binds p97 as well as the p97 adaptor Faf1 (Franz et al. atenation were all unaffected (Supplemental Fig. S2A–J). 2016). Despite this progress, critical questions remain un- Our results show that plasmid replication recapitulates answered: Which E3 ubiquitin ligase ubiquitylates Mcm7 Cullin RING ligase-dependent and p97-dependent CMG in higher eukaryotic cells? When is Mcm7 ubiquitylated unloading as seen on sperm chromatin (Moreno et al. and what triggers this event? How is Mcm7 ubiquityla- 2014) and in cells (Maric et al. 2014) and support our pre- tion coordinated with replisome disassembly? What is vious conclusion that CMG removal is not required for the nature of the p97 complex that promotes unloading? the DNA transactions underlying termination (Dewar To identify the vertebrate Mcm7 ubiquitin ligase, we et al.