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CMG Helicase and DNA Polymerase E Form a Functional 15-Subunit Holoenzyme for Eukaryotic Leading-Strand DNA Replication

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CMG Helicase and DNA Polymerase E Form a Functional 15-Subunit Holoenzyme for Eukaryotic Leading-Strand DNA Replication CMG helicase and DNA polymerase 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 replisome show polymerases (Pol) dedicated to bulk synthesis of the leading and that the leading and lagging strand replicases are coupled and lagging strands. Pol α/primase 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 proteins, 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 protein 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 replisomes 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 Escherichia coli, 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.
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