CMG and DNA e form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

Lance D. Langstona,b, Dan Zhanga,b, Olga Yurievaa,b, Roxana E. Georgescua,b, Jeff Finkelsteina,b, Nina Y. Yaoa,b, Chiara Indianic, and Mike E. O’Donnella,b,1

aThe Rockefeller University, bHoward Hughes Medical Institute, New York, NY 10065; and cManhattan College, Riverdale, NY 10471

Contributed by Mike E. O’Donnell, September 23, 2014 (sent for review August 13, 2014)

DNA replication in eukaryotes is asymmetric, with separate DNA Detailed biochemical studies of the E. coli show (Pol) dedicated to bulk synthesis of the leading and that the leading and lagging strand replicases are coupled and lagging strands. Pol α/ initiates primers on both strands that intimately linked to the replicative helicase, a feature also are extended by Pol e on the leading strand and by Pol δ on the common to the well-characterized T4 and T7 bacteriophage lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the replication systems (9–11). For this reason, it has been assumed leading strand and is proposed to recruit Pol e for leading-strand that the same would be true of eukaryotic systems, and this synthesis, but to date a direct interaction between CMG and Pol e notion has been strongly reinforced by the identification in has not been demonstrated. While purifying CMG helicase overex- yeast of replication progression complexes (RPCs), large pressed in yeast, we detected a functional complex between CMG multiprotein complexes containing, among other , e e CMG, Mcm10, Mrc1, and Ctf4 (12, 13). The RPC also contains and native Pol . Using pure CMG and Pol , we reconstituted a sta- α ble 15-subunit CMG–Pol e complex and showed that it is a functional Pol /primase under low-salt conditions, suggesting that it is more – weakly bound, and binding of Pol α to the replisome is abolished polymerase helicase on a model replication fork in vitro. On its – own, the Pol2 catalytic subunit of Pol e is inefficient in CMG-depen- in cells lacking Ctf4 or its metazoan counterpart, AND-1 (13 16). Ctf4 binds both the catalytic Pol1 subunit of Pol α andGINSin dent replication, but addition of the Dpb2 subunit of Pol e, yeast and thus is thought to tether Pol α to CMG in the replisome known to bind the Psf1 protein subunit of CMG, allows stable syn- δ (13, 16, 17). thesis with CMG. Dpb2 does not affect Pol function with CMG, and Neither Pol δ nor Pol e is found in the most highly purified thus we propose that the connection between Dpb2 and CMG helps e RPCs, which are defined by mass spectrometry of proteins bound to stabilize Pol on the leading strand as part of a 15-subunit lead- after sequential affinity purification of two separate CMG com- ing-strand holoenzyme we refer to as CMGE. Direct binding between ponents from a cell extract (12, 13). However, the noncatalytic e e Pol and CMG provides an explanation for specific targeting of Pol to Dpb2 protein subunit of Pol e is known to bind to the GINS the leading strand and provides clear mechanistic evidence for how component of CMG, and recent evidence suggests that this in- strand asymmetry is maintained in eukaryotes. teraction helps maintain Pol e at the replication fork (18, 19). Pol δ was shown to bind Pol α via its nonessential Pol32 subunit (20), DNA replication | replication fork | helicase | polymerase | CMG suggesting that Pol δ might be recruited from solution to extend primers initiated by Pol α/primase and may only associate eplisomes are multisubunit protein complexes that co- transiently with the core replisome. Rordinately unwind duplex DNA and duplicate both parental To study the eukaryotic replisome in detail, we initiated a strands during chromosomal replication. Detailed studies of long-term project to purify the numerous components of the cellular and viral systems show that the basic functional units of replication—helicase, primase, and DNA polymerase (Pol)—are Significance common to all whereas the evolutionary histories of the individual components in different kingdoms are distinctive and All cells must replicate their chromosomes prior to cell division. diverse (1). Accordingly, the sequence and structure of replisome This process is carried out by a collection of proteins, known as components are unrelated, and thus connections and coordination the replisome, that act together to unwind the double helix among the different functional units can be expected to vary widely. and synthesize two new DNA strands complementary to the The most well-studied cellular replisome to date, bacterial two parental strands. The details of replisome function have , uses multiple copies of a single DNA poly- been worked out for bacteria but are much less well un- merase to replicate both parental strands, and the action of these derstood for eukaryotic cells. We have developed a system for polymerases is coordinated by a multifunctional clamp loader studying eukaryotic replisome function in vitro using purified that also connects to the replicative helicase (2). For reasons that proteins. Using this system, we have identified a direct in- are still unclear, the eukaryotic replisome uses three different teraction between the component that unwinds the DNA, polymerases for normal chromosome duplication, including one the CMG (Cdc45-MCM-GINS) helicase, and the component for the leading strand (Pol e) and two for the lagging strand (Pol that replicates the leading strand, DNA polymerase e,to α/primase and Pol δ)(3–5). Similarly, whereas the replicative form a large helicase–polymerase holoenzyme comprising helicase in E. coli is a homohexamer of DnaB, the eukaryotic CMG 15 separate proteins. (Cdc45-MCM-GINS) helicase consists of 11 distinct subunits as- sembled on chromatin by loading of the heterohexameric Mcm2-7 Author contributions: L.D.L. and M.E.O. designed research; L.D.L., D.Z., R.E.G., N.Y.Y., and helicase core at an origin and its subsequent activation by associ- C.I. performed research; L.D.L., O.Y., R.E.G., and J.F. contributed new reagents/analytic ation with Cdc45 and the heterotetrameric GINS (Sld5-Psf1-Psf2- tools; L.D.L. and M.E.O. analyzed data; and L.D.L. and M.E.O. wrote the paper. Psf3) complex at the onset of S-phase to form the CMG complex The authors declare no conflict of interest. (6–8). Among other things, the complexity of the eukaryotic sys- Freely available online through the PNAS open access option. tem reflects the need to restrict chromosome duplication to a sin- 1To whom correspondence should be addressed. Email: [email protected]. gle round in a normal cell cycle so that proper ploidy can be This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. maintained across multiple chromosomes after cell division. 1073/pnas.1418334111/-/DCSupplemental.

15390–15395 | PNAS | October 28, 2014 | vol. 111 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1418334111 Downloaded by guest on October 1, 2021 RPC/replisome from the model eukaryote Saccharomyces cerevisiae. Thyroglobulin Pioneering work on Drosophila and human CMG showed that an A 670 kDa active helicase complex could be obtained by coexpression of all 11 fxn # 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Mcm 2-7 subunits in insect cells (7, 21) so we cooverexpressed all 11 CMG Cdc45 - subunits in yeast and purified the complex to homogeneity (22). We showed that, like its human counterpart, yeast CMG is capable of catalyzing replication of a model replication-fork substrate (21, 22). Sld5 - Using this system, we also showed that CMG enforces a preference Psf1/2 - for Pol e over Pol δ in leading-strand replication whereas pro- Psf3 - liferating cell nuclear antigen (PCNA) enforces the opposite pref- δ erence on the lagging strand (22). Preferential binding of Pol to B Superose 6 fraction number PCNA has been clearly demonstrated and provides an explanation * dT 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

60 Boiled No CMG δ dN31 for the dominance of Pol in lagging-strand synthesis (23), but the dT39 nature of any interaction between Pol e and CMG on the leading strand is poorly understood. dN30 While purifying CMG from yeast, we identified a direct in- * teraction between overexpressed CMG and native Pol e to form 30% a multifunctional eukaryotic leading-strand holoenzyme that we 20% refer to as CMGE. Using separately purified CMG and Pol e,we reconstituted a stable, 15-subunit CMGE and showed that it is an 10% active helicase–polymerase in vitro. We also show that the Dpb2 subunit of Pol e, which binds to the Psf1 protein subunit of 0% GINS, promotes efficient Pol e function with CMG. Direct % substrate unwound 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 binding of Pol e to the full CMG complex has not been pre- C D dC viously demonstrated, and this interaction provides a mechanis- dT * 30 + CMG * 80 + CMG dN39 dT25 dN Boil tic foundation for preferential replication of the leading strand 70 No CMG e – by Pol as part of a stable helicase polymerase holoenzyme (4). M13 200-mer 7.3 kb circle

Results BIOCHEMISTRY Purified Yeast CMG Is an Active Helicase. As part of our efforts to * * reconstitute an active eukaryotic replisome from separately pu- % unwound 0% 3% 14% 36% % unwound 0% 0% 0% rified components, we cooverexpressed all 11 subunits of the S. cerevisiae Fig. 1. Gel-filtration analysis of purified CMG. The 11-subunit CMG overex- CMG helicase in yeast cells and purified the full CMG pressed in yeast was purified through two affinity columns, and 300 μL of purified complex using separate tags on Mcm5 and Sld5 or Cdc45 and protein was injected onto a Superose 6 column. (A) Indicated fractions from the Mcm5, similar to the approach used to purify the Drosophila and Superose 6 column elution were separated on a 10% SDS/PAGE gel and stained human CMG complexes from insect cells (7, 21, 22). Gel filtra- with Coomassie Blue. (B) Two microliters of each CMG fraction from A was assayed tion of the resulting CMG produced a highly pure complex with for helicase activity in a 12-μLreactionusinga5′-radiolabeled fork substrate (see a peak at the expected position for an ∼785-kDa complex (Fig. schematic at Left) for 20 min at 30 °C in the presence of 2 mM ATP. Products were 1A). The ability of CMG to unwind a model Y-substrate was separated on a 10% native PAGE gel and subjected to phosphorimagery (Top). The tested, and the peak of unwinding activity comigrated with the chart below shows the percent of substrate unwound by each indicated CMG peak of CMG (Fig. 1B, fraction 23), confirming that CMG forms fraction as determined by quantitative analysis of the phosphorimage. (C)Anoligo a functional helicase complex as previously observed (7, 21). was radiolabeled at its 5′ tail and annealed to M13mp18 circular ssDNA (see schematic at Left). The M13 substrate (0.5 nM) was incubated with 12 nM CMG and μ CMG Helicase Is Active on Large but Not Small Circular DNA Substrates. 1 mM ATP at 30 °C, and 12- L aliquots were removed at the indicated times and Previous work in bacteria and phages has shown that rolling-circle analyzed as in B. The percentage of the substrate unwound by CMG is indicated below the phosphorimage. (D) A circular DNA of 200 nt was constructed as de- replication is an ideal system for studying the action of replisomes, ′ and indeed a previous report demonstrated rolling-circle replica- scribed in SI Materials and Methods.Anoligowitha5 dC30 tail and 70 nt com- plementary to the circle was radiolabeled at its 5′ end and annealed to the circle, tion supported by human CMG and Pol e (21). Earlier studies which contains a dT25 region adjacent to the annealed duplex to facilitate CMG showed that the Drosophila and human CMG complexes can un- μ ′ loading (21). The substrate was incubated with CMG and ATP as in C,and12- L wind a short 5 -tailed oligo annealed to 7.3 kb circular bacterio- aliquots were stopped at the indicated times. phage M13mp18 ssDNA and that yeast CMG also has this property, as shown in Fig. 1C. The substrates used for rolling-circle replication are much smaller than M13, typically in the 100- to 200- CMG Functions in Leading-Strand Replication with Pol e. The inability nt range, and indeed a 200-nt circle was used for leading-strand of CMG to unwind small circular substrates led us to construct a linear replication in the human system although CMG-unwinding activity forked DNA substrate for replication, as reported previously (22). was not directly demonstrated on this substrate (21). Leading-strand replication on a forked linear DNA can be monitored To determine whether yeast CMG can load efficiently and pro- by following extension of a radiolabeled primer annealed to the forked ductively onto small circular , we annealed a 70-nt oligo with e ′ substrate, and the function of CMG with Pol over a 60-min time a radiolabeled 5 dC30 tail to a 200-nt circle similar to that used for course is shown in Fig. 2. As shown in the scheme at the top, CMG is replication in the human system (21) and monitored unwinding of preincubated with the 2.8-kb forked linear DNA substrate in the ab- the labeled oligo by CMG during a 40-min time course. In contrast sence of ATP for 10 min to permit CMG loading, replication is initi- to the M13 circle, we observed no unwinding by CMG on the 200-nt e circle (Fig. 1D) or on a larger 300-nt version of the same circle (Fig. ated by adding Pol , replication factor C (RFC), PCNA, and dNTPs, S1A). As a control for unwinding by CMG in this sequence context, and the reaction is started by adding ATP to fuel the CMG helicase. we assembled a linear fork substrate with the same sequence as the (RPA) is added after initiation because it was previously shown to inhibit CMG loading in vitro (22). Control circular forks by annealing the radiolabeled 5′ dC30–tailed 70-mer to a linear oligo used to construct the small circular substrates. As experiments showed that neither Pol e nor CMG on its own was ca- shown in Fig. S1B, this linear substrate is progressively unwound by pable of leading-strand synthesis on this substrate (Fig. S2A) whereas CMG over the course of 40 min. Taken together, the simplest ex- CMG and Pol e cooperatively extended the leading strand to full planation of these results is that yeast CMG loads poorly onto small length (Fig. 2 and Fig. S2A). CMG did not stimulate synthesis by Pol e circular substrates, at least under the conditions used in our assays. on a singly primed ssDNA template (Fig. S3) so we inferred that CMG

Langston et al. PNAS | October 28, 2014 | vol. 111 | no. 43 | 15391 Downloaded by guest on October 1, 2021 1) LOAD 2) INITIATE DNA 3) START DNA leading strand with Pol e, we sought to understand the underlying CMG SYNTHESIS UNWINDING mechanism whereby CMG exhibits a preference for Pol e over Pol CMG + Pol + STOP δ in leading-strand synthesis as demonstrated in both the yeast and + ATP 2.8 kb forked + RFC/PCNA at t human systems (21, 22). While purifying the CMG complex, we linear DNA + dNTPs RPA frequently observed two unidentified proteins coeluting with CMG * * * through two affinity columns (Fig. 3A). These proteins were not observed in preparations of overexpressed Mcm2-7 under similar Lane # 1 2 3 4 5 6 7 8 conditions and thus are specific to CMG (Fig. S5). The copurifying proteins could be removed by extensive washing or further puri- 2.8 kb fication (Fig. 1A), but their persistence through two purification full length extension steps under a variety of conditions was noteworthy. Furthermore, one of these additional proteins was much larger than any of the 1.5 kb - CMG subunits, suggesting that it might be a native yeast protein copurifying with CMG. To investigate the identity of these pro- 1.0 kb - teins, we analyzed the gel in Fig. 3A by mass spectrometry and identified the two copurifying proteins as Pol2 and Dpb2, the two essential subunits of the Pol e complex (30, 31). Although we an- 0.5 kb - alyzed only the visible Pol 2 and Dpb2 bands, we presumed that the small Dpb3/4 subunits of Pol e were also present. Identification of a stable CMG–Pol e complex was unexpected given that Pol e was not previously detected in replication progression complexes after Starting double-affinity purification (12, 13). substrate To determine whether this fortuitously purified CMG-Pol e complex was functional, we used the linear replication fork assay described in Fig. 2 to monitor leading-strand replication by – e e ′– ′ Fig. 2. Time course of leading-strand replication by CMG plus Pol e.The CMG Pol . Because CMG-bound Pol has a 3 5 proofread- μ ing exonuclease activity, we added two dNTPs during the initial reaction scheme is summarized above the gel. In a 180- L reaction, 25 e nM CMG was incubated 10 min at 30 °C with 1 nM 2.8 kb forked DNA CMG-loading step to prevent Pol from completely digesting substrate with a 5′-radiolabeled leading-strand primer followed by the primer or extending it. As shown in Fig. 3B, the CMG–Pol e addition of 6 nM RFC, 20 nM PCNA, and 15 nM Pol e and 60 μM dNTPs. complex purified directly from yeast extended the radiolabeled After 5 min, the reaction was started with 4 mM ATP followed 1 min later by addition of 350 nM RPA. The 20-μL aliquots of the reaction were stopped at the indicated times after addition of ATP and analyzed in a denaturing polyacrylamide gel. Positions of radiolabeled molecular A Pol2 B weight reference bands are shown to the left of the gel and to the right of the gel are lane profiles of the indicated time points generated by ImageQuant analysis of the scanned phosphorimage. The percentage of Mcm6 Complete Mcm2/3 leading strand primers extended past the forked junction at each time point are shown Mcm4/7 extension in Fig. S2B. Mcm5 Dpb2 CMG-Pol + Cdc45 2.8 kb DNA + 1.5 kb was acting through its helicase activity to unwind the fork rather than dCTP/dGTP e directly stimulating the polymerase activity of Pol . 1.0 kb As shown in the lane profiles to the right of Fig. 2 and in the B graph in Fig. S2 , full-length 2.8-kb products appeared as early as + RFC/PCNA the 7-min time point, indicating that a subpopulation of leading- a dATP/dTTP strand replisomes was capable of replicating at an average rate of * 0.5 kb ∼400 bp/min, within four- to sevenfold of the in vivo rate (24, 25). However, most replisomes required 20–60 min after addition of Sld5 ATP to form full-length products. This rate was ∼10- to 20-fold + ATP slower than the typical estimates for replication-fork progression SSB in yeast but was comparable to the rate of replication using Starting S-phase yeast extracts on linear and circular plasmid substrates STOP substrate – Psf1** at t (26 28). We presume that additional factors or posttranslational Psf2 modifications not present in these reactions may increase the Psf3 speed of the minimal replisome in vitro. We also note that the present reactions do not include Pol α/primase or Pol δ, the po- lymerases responsible for lagging-strand synthesis. In the absence Fig. 3. Identification of a Functional CMG–Pol e complex. (A) Native Pol e of lagging-strand synthesis, ssDNA accumulates on the lagging copurifies with overexpressed CMG. CMG was purified through two affinity strand, and this excess ssDNA may interfere with helicase pro- columns (FLAG-Mcm5 and GST-Sld5), and the GST tag was cleaved. A sample gression, perhaps by binding to Cdc45 as part of the replication of purified protein was separated by SDS/PAGE and stained with Coomassie stress response as suggested (29). Indeed, we observed that Blue. Indicated bands were analyzed by mass spectrometry, and the protein(s) leading-strand replication by CMG-Pol e on our model fork identified for each band is shown to the left (CMG) and right (Pol e subunits) E. coli of the gel. The bands labeled * and ** are the Prescission Protease and the substrates was modestly stimulated by RPA (22) and by e single-strand DNA binding protein (SSB) (Fig. S4), both of cleaved GST tag, respectively. Copurification of functional native Pol was observed in nine separate purifications of CMG. (B) Leading-strand replica- whichbindtossDNAandperhapsalleviatetheinhibitionof tion by 30 nM CMG with copurifying Pol e. The scheme of the leading strand helicase progression. replication reaction is in the gray box (Left). Reactions contained 2 nM RFC, 10 nM PCNA, and 5 mM ATP, and 200 nM E. coli SSB was used instead of RPA Identification of a Functional Complex Between Overexpressed CMG (21). Positions of radiolabeled molecular weight reference bands are shown and Native Pol e. Having demonstrated that yeast CMG is a func- to the left of the gel (Right) and lane scans of the indicated time points are tional helicase that can cooperatively unwind and synthesize the shown to the right of the gel.

15392 | www.pnas.org/cgi/doi/10.1073/pnas.1418334111 Langston et al. Downloaded by guest on October 1, 2021 leading-strand primer to full length after 30 min with some com- CMG + DNA STOP + ATP plexes reaching full length after only 10 min. Examination of the Pol2 + Dpb2 at t lane profiles to the right of the gel in Fig. 3B shows that leading- + RFC/PCNA + dNTPs RPA [Dpb2] strand synthesis by the directly purified CMG–Pol e proceeded 400 25 nM with very similar kinetics to the reaction in Fig. 2 using separately 300 12.5 nM purified CMG and Pol e. These data provide strong evidence for aleading-strandCMG–Pol e holoenzyme we refer to as CMGE. 200 5 nM 100 0 nM – e Relative intensity Reconstitution of CMGE, a Multifunctional CMG Pol Holoenzyme. To 0 further investigate complex formation between CMG and Pol e, 0.5 kb 1.0 1.5 2.0 3.0 kb we analyzed the migration of Pol e during ultracentrifugation in 5 nM Pol2 a glycerol gradient in the presence and absence of CMG. The four- [Dpb2] nM 0 5 12.5 25 Pol subunit Pol e complex is ∼380 kDa and CMG is ∼785 kDa so, if Pol e binds to CMG, then the reconstituted complex should mi- 3.0 kb grate as a much larger species, ∼1.15 mDa, causing a clear shift in the migration of Pol e compared with the no-CMG control. As 2.0 kb shown in Fig. 4, Pol e alone migrated as a single species with a peak around fraction 32 (middle gel). When mixed with CMG, however, 1.5 kb e a portion of Pol shifted its migration to a much higher molecular 1.0 kb weight, peaking at fraction 14, whereas excess Pol e (not bound to CMG) migrated at the position expected for Pol e alone (Fig. 4, top gel). These results demonstrate the formation of a specific 0.5 kb complex between Pol e and CMG with no other protein required to Lane 1 2 3 4 5 6 7 8 9 10 mediate the interaction. They also confirm, as shown in the ex- Fig. 5. Dpb2 links Pol2 to CMG for CMGE function. Leading-strand repli- panded view of CMGE fraction 14 at the right of Fig. 4, that the e e cation reactions are similar to Fig. 2 but using either Pol2 or Pol . As shown Dpb3 and Dpb4 subunits of Pol are incorporated into a CMGE in the reaction scheme (Top), CMG was mixed with 1.0 nM DNA template holoenzyme comprising 15 separate subunits. Densitometric separately from 5 nM Pol2 and Dpb2 (lanes 1–8) or 20 nM Pol e (lanes 9 and analysis of Pol2/Dpb2 and Mcm2-7/Cdc45 from the CMGE com- 10). Both mixtures were preincubated for 10 min and then combined for – e plex peak (fractions 12 16 in Fig. 4) indicated that Pol and CMG 5 min. The reaction was started with ATP, stopped at the indicated times, BIOCHEMISTRY were present in equimolar amounts in the CMGE complex and processed as in Fig. 2. (Middle) The graph shows ImageQuant lane scans (Fig. S6A). corresponding to 30 min reaction time at different Dpb2 concentrations To determine whether the reconstituted CMGE was func- with 5 nM Pol2. tional for leading-strand replication, we used an assay similar to that described in Fig. 2. to test the peak fraction of CMGE from a glycerol gradient. In the absence of added Pol e, reconstituted CMGE was able to unwind the duplex and synthesize the leading strand to full length (Fig. S6B). Together with the data from Fig. 3, these results indicate that CMGE is a functional holoenzyme with both unwinding and DNA polymerase functions. 670 kDa 158 kDa 50 kDa CMGE Fraction14 - Pol2 - Pol2 The Dpb2 Subunit of Pol e Is Required for Efficient Leading-Strand Mcm2-7 - Dpb2 e \ Cdc45 Replication with CMG. The Dpb2 subunit of Pol binds to both the - Mcm6 - Mcm3 catalytic subunit Pol2 and to the Psf1 subunit of GINS and, - Mcm2/4 - Sld5 - Mcm7 - Mcm5 whereas this connection has been shown to be required to recruit - Dpb3 - Dpb4 - Dpb2 Pol e to the replication fork, it was not clear whether it is essential - Psf1/2 - Cdc45 - Psf3 for ongoing Pol e function with CMG during elongation (19, 30, - Pol2 32, 33). Indeed, a three-subunit form of Pol e lacking Dpb2 was - Dpb2 shown to be a fully functional and processive polymerase on singly primed circular substrates so Dpb2 is not required for the polymerase activity of Pol e (34, 35). To investigate the role of - Sld5 - Dpb3 Dpb2 in Pol e function with CMG at a replication fork, we sep- - Dpb4 arately purified Dpb2 and the Pol2 catalytic subunit (Fig. S7)and Mcm2-7 - Dpb3 - Cdc45 examined Pol2 activity in leading-strand replication assays with - Dpb4 CMG in the presence and absence of Dpb2 (30, 36). As shown in Fig. 5, in the absence of Dpb2, Pol2 gave a weak full-length signal - Psf1/2 after 30 min (lane 2). Upon addition of increasing amounts of - Psf3 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 fxn# Dpb2, however, the ability of Pol2 to synthesize the full 2.8-kb sedimentation leading strand was restored (Fig. 5, compare lanes 4, 6, and 8 with Fig. 4. Reconstitution of a Stable CMG–Pol e Complex. Glycerol gradient lane 2; also see plots above the gel), suggesting that the con- elution profiles of CMG alone (Bottom), Pol e alone (Middle), and a mixture nection between Pol2 and CMG via Dpb2 is important for effi- of Pol e and CMG (Top). Fractions were collected from the bottom of the cient Pol e function in leading-strand replication with CMG. centrifuge tubes so that the largest molecular weight species were collected The foregoing experiments do not exclude the possibility that first, and a sample of each indicated fraction was separated by SDS/PAGE Dpb2 is a general stimulatory factor for CMG so, to determine and stained with Coomassie Blue. The elution peak of each complex (as whether this effect is specific to Pol2/Pol e, we added Dpb2 to determined by densitometry shown in Fig. S6A) is indicated in the gels. δ γ leading-strand replication assays containing Pol /CMG. Our Molecular mass size standards (thyroglobulin 670 kDa, -globulin 158 kDa, previous results showed that Pol δ is not able to replicate the and ovalbumin 44 kDa) were run in a parallel gradient, and the migration substrate to full length with CMG under any conditions tested peaks of each standard are indicated at the top of the gels. (Right)Anex- δ panded view of CMGE Fraction 14 showing the presence of all 15 CMGE (22), and indeed addition of Dpb2 had no effect on Pol function subunits. The migration of each subunit is indicated to the right of the gels. with CMG (Fig. S8). Taken together, these results strongly suggest Reconstitution of the CMG–Pol e complex was demonstrated by glycerol that Dpb2 is a specific factor linking Pol e to CMG in the leading- gradient a total of six times. strand CMGE holoenzyme and that this connection is important

Langston et al. PNAS | October 28, 2014 | vol. 111 | no. 43 | 15393 Downloaded by guest on October 1, 2021 not just for recruitment of Pol e to origins but also for elongation we show that leading-strand replication by Pol e absolutely during S-phase. requires CMG (Fig. S2A) and is stimulated by RPA, yet loading of CMG onto DNA is inhibited by RPA (22). This inhibition of Discussion helicase loading is reminiscent of the E. coli system, where Pol e is recruited to prereplicative complexes along with GINS as loading of the replicative helicase DnaB is strongly inhibited by part of a complex that is essential for CMG formation, and sub- SSB (38). Thus, if CMG were to fall off the DNA, it would be sequent activation of CMG is the defining step in replication unlikely to rebind in the presence of RPA so we propose that CMG initiation (32, 33, 37). The Dpb2 subunit of Pol e also binds to is loaded onto the forked DNA substrate before addition of RPA GINS, and this connection is essential for Pol e recruitment to and remains bound until replication is complete. origins (32), but the mechanism by which Pol e is specified for The connection between Pol e and CMG via Dpb2 contributes ongoing leading-strand synthesis is poorly understood. We have to the stability of the binary complex during replication (Fig. 5), identified a stable complex between CMG helicase and Pol e to but, given that both Pol e and CMG bind to DNA, the contribu- form a 15-subunit holoenzyme, CMGE, that is functional for tion of the individual complexes to the stability of the holoenzyme leading-strand replication in vitro (Figs. 3 and 4). CMGE con- during active replication remains to be determined. Although Pol currently unwinds and synthesizes DNA on the leading strand, δ does not bind DNA in the absence of the PCNA clamp, Pol e and the tight connection between helicase and polymerase binds DNA on its own with a preference for primed template (23, explains how Pol e is specified for leading-strand replication over 39, 40). It will be interesting to investigate whether DNA binding Pol δ and other polymerases both in vivo and in vitro (4, 21, 22). by Pol e helps to stabilize CMG on DNA, in which case the sta- The persistence of a functional CMG–Pol e complex during bility of the active CMGE holoenzyme may be greater than that of purification through two highly selective affinity columns under its individual components. high ionic strength conditions (Fig. 3) was unexpected for several Studies of leading-strand replication by human Pol e showed reasons. Most significantly, Pol e does not copurify with isolated fivefold stimulation by addition of CMG, but, unlike the yeast yeast replication progression complexes (RPCs) after two affinity system, replication did not absolutely require CMG (21). Further- columns (12, 13). Indeed, Pol α can be isolated with purified more, although RPA stimulated the of unwinding by RPCs provided low ionic strength is used, yet Pol e is not ob- human CMG, it did not support CMG-dependent leading-strand served even in these mild isolation procedures (13). Furthermore, replication on a rolling circle by human Pol e, which was dependent mixing of S-phase yeast extracts containing tagged and untagged instead on E. coli SSB (21). We did not observe helicase activity by versions of Dpb2 followed by immunoprecipitation of CMG led yeast CMG on rolling-circle substrates (Fig. 1D), and we also show to the conclusion that Pol e binding at the replication fork was that leading-strand replication by CMG–Pol e on linear fork sub- highly dynamic, implying that Pol e does not stably associate with strates occurs in the absence of single strand-binding proteins and CMG (32). Finally, the C-terminal domain of the Psf1 subunit of is modestly stimulated by both RPA and SSB (Fig. S4) (22). It is GINS binds to Pol e, yet a study of Drosophila CMG indicated unclear whether these differences reflect a true divergence between that the C-terminal domain of Psf1 is essential for CMG forma- the yeast and human systems or simple discrepancies between assay tion and was putatively assigned to interaction with Mcm5 and systems and conditions. potentially unavailable to bind other proteins (8). We do not yet know the processivity of Pol e in our reactions, Detailed time courses of CMG-dependent leading-strand rep- but the connection between Pol e and CMG may tether the lication by Pol e show that full-length 2.8-kb products are observed polymerase to the replisome so that, even if it falls off the primer after 7 min and accumulate steadily out to 30 min and beyond terminus, it can quickly rebind and continue synthesis. One po- (Figs. 2 and 3) (22). Along with our previously published results, tential drawback to a stable connection between Pol e and CMG is the need for dynamic exchange between high-fidelity and low- fidelity polymerases to overcome template blocks during fork progression (41). Temperature-sensitive mutants of the Dpb2 GINS subunit of Pol e that no longer bind stably to the Pol2 catalytic (i) Pol encounters Mcm2-7 subunit confer a strong mutator phenotype that is partially de- lesion pendent on Pol ζ, suggesting that low-fidelity polymerases have Leading strand lesion greater access to the leading strand when the CMG-Dpb2-Pol2 PCNA Pol connection is disrupted (34, 42, 43). Together, these data suggest a model whereby the Pol e–CMG connection is flexible, enabling translesion and other polymerases to bind the DNA temporarily (ii) Polymerase e switch without displacing Pol from the replisome. As illustrated in Fig. 6, we propose that the proximity of Pol e tethered to the fork by TLS Pol CMG serves to limit the action of the low-fidelity polymerase and enable rapid recovery of the primer terminus by the high- fidelity Pol e as soon as the lesion is bypassed. This hypothesis and the dynamics of processes that connect the leading-strand (iii) Lesion bypass apparatus to lagging-strand enzymes remain exciting avenues for future exploration. Materials and Methods (iv) Pol resumes Experimental procedures are described in full in SI Materials and Methods.

CMG Expression and Purification. Yeast strains expressing all 11 CMG subunits Fig. 6. Hypothesis of Pol e retention at a fork by binding CMG. Binding of Pol e were induced with galactose, harvested, and frozen as pellets in liquid ni- (green) to GINS (purple) in CMG may help retain Pol e at the fork upon dissoci- trogen. Extracts made from grinding frozen pellets were purified on two ation from the primer terminus. One implication of this action is to facilitate successive affinity columns (22) and further purified on Superose 6 polymerase switching at template lesions, illustrated in the example shown: (i) where indicated. All oligonucleotides are given in Table S1. CMGE approaches a lesion (red octagon) in the leading strand template and (ii) Pol e vacates PCNA (red) and the primer terminus upon encountering the lesion Helicase Assays. Reactions containing 0.5 nM radiolabeled DNA substrate but remains bound to CMG, allowing access to a translesion synthesis (TLS) were incubated at 30 °C, and products were separated on 10% (wt/vol) polymerase (pink). (iii) The TLS Pol(s) bypasses the lesion whereupon (iv)Pole Native PAGE minigels. Amounts of added CMG helicase and incubation rebinds the primer terminus and resumes high-fidelity replication. conditions are indicated in the figure legends.

15394 | www.pnas.org/cgi/doi/10.1073/pnas.1418334111 Langston et al. Downloaded by guest on October 1, 2021 Replication Assays. Reactions contained 1.0 nM pUC19 primed fork with a Reconstitution of CMGE. To examine CMG–Pol e complex formation, 320 pmol 5′-radiolabeled leading-strand primer annealed to the fork. Reaction vol- of CMG was mixed with an excess of Pol e (480 pmol) for 30 min and sep- umes, ATP added, proteins added, and their amounts are indicated in the arated in a 15–35% glycerol gradient at 4 °C for 18 h at 260,000 × g. Frac- ∼ μ figure legends. Unless otherwise noted, CMG was first incubated at 30 °C tions (7 drops, 170 L) were collected from the bottom of the centrifuge μ with the substrate for 10 min. Pol e was added along with RFC/PCNA and 60 tubes, and 20- L samples were analyzed by SDS/PAGE and stained with Coomassie Blue. Identical gradient analyses were performed for CMG and μM dNTPs and incubated a further 5 min to extend the primer into a short Pol e alone. Gels were scanned in an ImageQuant LAS4000 (GE Healthcare) ssDNA gap at the fork where CMG is expected to load. ATP was then added and analyzed using ImageQuant TL v2005 software. to initiate unwinding by CMG, and SSB or RPA was added last. At the in- dicated times after addition of ATP, aliquots of the reaction were stopped ACKNOWLEDGMENTS. We thank the Rockefeller Proteomics Resource and separated on an alkaline agarose gel. Exceptions to this protocol are Center for mass spectrometry. We are grateful for support from NIH Grant indicated in the figure legends. GM38839 and the Howard Hughes Medical Institute.

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