Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork

Abstract

Eukaryotes use distinct polymerases for leading- and lagging-strand replication, but how they target their respective strands is uncertain. We reconstituted Saccharomyces cerevisiae replication forks and found that CMG helicase selects polymerase (Pol) ɛ to the exclusion of Pol δ on the leading strand. Even if Pol δ assembles on the leading strand, Pol ɛ rapidly replaces it. Pol δ-PCNA is distributive with CMG, in contrast to its high stability on primed ssDNA. Hence CMG will not stabilize Pol δ, instead leaving the leading strand accessible for Pol ɛ and stabilizing Pol ɛ. Comparison of Pol ɛ and Pol δ on a lagging-strand model DNA reveals the opposite. Pol δ dominates over excess Pol ɛ on PCNA-primed ssDNA. Thus, PCNA strongly favors Pol δ over Pol ɛ on the lagging strand, but CMG over-rides and flips this balance in favor of Pol ɛ on the leading strand.

Figure 1

Reconstitution of a leading-strand replisome with CMG, Pol ε, RFC, PCNA and RPA. (a) Scheme of the assay. The 3.2-kb forked DNA substrate was constructed by ligation of a synthetic primed replication fork to a 3.2-kb duplex created with nucleotide bias to enable specific labeling of the leading strand with [³²P]dCTP. CMG was assembled on the DNA with AMP-PNP in a 10-min preincubation; then Pol ε, PCNA and RFC were added for 2 min along with cold dATP and dGTP before replication was initiated upon addition of RPA, ATP, dTTP and [³²P]dCTP. Experimental details and protein concentrations are in Online Methods. (b) Alkaline agarose gel of reaction products. Lanes 1–5, time course of the full reaction; lanes 6–9, reactions with indicated components omitted. Arrows indicate the peak lengths of DNA product at 2 and 4 min. Quantitation of the results is in Supplementary Figure 2. MW, molecular weight.

Figure 2

Comparison of Pol ε and Pol δ in leading-strand replication with CMG. Time courses of leading-strand replication with either Pol ε (a) or Pol δ (b). Reactions were staged as illustrated in the reaction scheme (Fig. 1a), except the leading-strand primer was 5′-end-labeled with ³²P instead of with [³²P]dNTPs, and linearized 2.8-kb pUC19 was used as the duplex region. RFC/PCNA denotes addition of both components. The right lanes of each gel (lanes 6–8 in a and 6–9 in b) show the results of omitting individual components as indicated. The letters a–c at right of gel in a mark the full-length DNA position, the primer-extension product to the forked junction and the unextended ³²P primer, respectively. The graphs to the right are ImageQuant analysis (in arbitrary units) of lanes 1–5 (a) and 3–5 (b), with time (min) indicated, and the peaks of the fits correspond to the average length of DNA. Scans were analyzed by fitting to a double (a) or single (b) Gaussian distribution (black lines). The uncropped gel images are shown in Supplementary Figure 7.

Figure 3

CMG selects Pol ε from a mixture of Pol ε and Pol δ. (a) Top, scheme of the assay. Reactions were performed as in Figure 2, except a mixture of Pol ε and Pol δ were added along with PCNA and RFC for 2 min before initiation of DNA synthesis. Reactions were quenched after 6 and 20 min (′). Bottom, alkaline agarose gel of the reaction products. Concentrations of each polymerase are indicated above the gels. (b) Top, scheme of the assay. CMG was assembled on the ³²P-primed forked DNA; then either 10 nM Pol ε or 10 nM Pol δ was added along with RFC and PCNA. Replication was initiated and continued 4 min before addition of the second DNA polymerase—either Pol δ or Pol ε as indicated. Replication was quenched after a further 16 min. Bottom, alkaline agarose gel of products. The order of polymerase addition and the concentration of the second polymerase are indicated above each lane. The numbers at the bottom of each gel represent relative percentage of full-length product in each series of reactions (top row) and total percentage of substrate extended past the forked junction (bottom row).

Figure 4

Pol ε functions stably with CMG, whereas Pol δ does not. (a,b) Leading-strand (ld) replication reactions as described in Figure 2 but with different amounts of either Pol ε (a) or Pol δ (b). For each reaction, 5-min and 20-min time points were analyzed. The last two lanes of a show the effect of omitting RFC and PCNA. In b, arrows indicate the products at the 20-min time point; the small ³²P primers were run off the end of the gel. The graphs to the right of each panel are ImageQuant stacked lane scans corresponding to 20-min reaction time at different polymerase concentrations. Scans were fit to single Gaussian distributions (black lines). The error to the fit is noted. The uncropped gel images are in Supplementary Figure 7.

Figure 5

Pol ε and Pol δ rapidly associate with a PCNA-primed template. (a) Scheme of the assay and the expected products. The primed ϕX174 ssDNA is coated with a SSB protein and preincubated with RFC and PCNA to load the clamp on the primed site. Polymerase (Pol ε or δ) is either included in the preincubation (top path) or is added after initiating synthesis (bottom path). Time courses of reactions with either Pol ε (b) or Pol δ (c) and either E. coli SSB (eSSB) or RPA are as indicated above the alkaline gels. Preinc, preincubation; std, standard. Quantitation of the results is in Supplementary Figure 4, and the uncropped gel image is in Supplementary Figure 7.

Figure 6

Pol δ is dominant over Pol ε on a lagging-strand model template. (a) Scheme of the assay and expected products for reactions with a mixture of polymerases. Polymerases were incubated together or alone with DNA along with RFC and PCNA, and then replication was initiated. Expected products, full length or incomplete, are illustrated at right. Reactions were performed as described in Online Methods. (b) Alkaline gel of DNA products from reactions with either 20 nM Pol ε alone (lanes 1–6), 5 nM Pol δ alone (lanes 7–12) or a mixture of 5 nM Pol δ and 20 nM Pol ε (lanes 13–18). Pol ε and Pol δ were premixed and incubated together with the DNA substrate before initiation of replication. (c) Lane scans of reaction products at 5 min. The inset shows the amount of DNA synthesis at the indicated times as determined by the area underneath the lane scans determined by multipeak (Gaussian distribution) analysis. The error bars represent the standard error of the fit.

Figure 7

Scheme of asymmetric polymerase assembly at a replication fork. (a) CMG recruits Pol ε to the leading strand and stabilizes it on DNA. If Pol δ associates with the leading strand, CMG does not stabilize it, and Pol δ dissociates. (b) PCNA strongly favors association of Pol δ over Pol ε on the lagging strand. (c) Replication fork with asymmetric distribution of DNA polymerases. Pol ε may bind CMG via contact between the Dbp2 subunit of Pol ε and the Psf1 subunit of CMG.