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.
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Acknowledgements
The authors are grateful for support from the US National Institutes of Health (GM38839) and Howard Hughes Medical Institute to M.E.O. The authors thank A. Tackett and B. Chait (Rockefeller University) for yeast strain OY001.
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R.E.G., L.L. and M.E.O. conceived the project and designed the experiments. R.E.G., L.L., N.Y.Y. and T.A. executed the experiments and analyzed the results together with M.E.O. L.D.L., O.Y. and J.F. designed the yeast strain constructions, and O.Y. and J.F. carried them out. R.E.G., L.D.L., O.Y. and D.Z. carried out protein purifications. M.E.O. and R.E.G. wrote the manuscript with input from L.D.L.
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Integrated supplementary information
Supplementary Figure 1 SDS-PAGE of CMG and helicase activity.
(a) Purified CMG (4 μg) was analyzed in a 8% SDS polyacrylamide gel. (b) The bar graph indicates the stoichiometry of Mcm2-7, Cdc45 and GINS subunits within the CMG prep (MCM2-7 is set at 1.0), as determined by comparing their intensities with protein standards of known amounts of each of these complexes determined by Bradford stain and loaded in the same gel. (c) Helicase activity of CMG was determined using an end-labeled forked DNA substrate explained in Extended Experimental Procedures. The 144 μl helicase assay contained 1.9 pmol CMG and 72 fmol radiolabeled fork. Reactions were performed at 30 °C in the presence of 2 mM ATP. The 27% unwinding observed using this 26-fold excess of yeast CMG over DNA is comparable to the 24% unwinding observed using a 40-fold excess of Drosophila CMG over a similar DNA substrate (and in similar conditions). Thus the yeast CMG displays comparable specific activity to Drosophila CMG (Ilves, I., Petojevic, T., Pesavento, J.J. & Botchan, M.R. Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. Mol Cell 37, 247-58 (2010)).
Supplementary Figure 2 Analysis of 3-kb forked DNA-replication products by CMG–Pol ɛ.
Reactions were performed using α − 32P-dCTP incorporation, and therefore the length of DNA products were corrected for the increase in 32P-dCMP incorporation with DNA length as in Kurth, I., Georgescu, R.E. & O'Donnell, M.E. A solution to release twisted DNA during chromosome replication by coupled DNA polymerases. Nature 496, 119-22 (2013). The analysis corresponds to the experiment of Fig. 1. Scans of 2′ (a) and 4′ (b) replication reactions are plotted at the same scale. The lane profiles were normalized to the corresponding molecular weight at each pixel in order to correct for the fact that longer products incorporate more radiolabel. (c) Normalized lane profiles for the 8′, 12′ and 20′ reactions; the inset is a plot of the total DNA synthesis (area under the scans) determined by fitting the data to a double Gaussian distribution (the error bars represent the standard errors of parameters obtained from the fitting). The dashed line denotes the end-labeled product due to polymerase/exonuclease idling activity of Pol ɛ (reactions missing CMG and ATP). (d) Lane profiles of replication reactions in which RFC/PCNA or RPA are omitted. The inset histogram shows the relative percentage of the accumulated 3kb product in the complete replication reaction versus the incomplete reactions.
Supplementary Figure 3 RPA inhibits fork unwinding if it is added before CMG.
Scheme of the unwinding reactions; CMG is either: (a) preincubated with the DNA fork for 10 min at 30 °C before adding RPA, or (b) added to the DNA fork after RPA. The unwinding reaction is initiated upon the addition of 1 mM ATP. (c) RPA inhibits binding but not unwinding by CMG. Native PAGE depicting time course reactions (2’ 10’ and 20’) performed as illustrated in the reaction schemes and described in Extended Experimental Procedures. In separate 55 μl reactions, 0.7 pmol CMG was pre-incubated with 27.5 fmol DNA at 30° for 20’ in the presence or absence of 0.55 pmol RPA. Unwinding was initiated by addition of ATP to 1 mM (and RPA where indicated) and 12 μl aliquots were removed at the indicated times after addition of ATP for analysis by native PAGE.
Supplementary Figure 4 Analysis of φX174 ssDNA replication products.
The analysis corresponds to the experiment of Fig. 5. Primed φX174 ssDNA is coated with a single-strand binding protein (E.coli SSB or S. cerevisiae RPA) and preincubated with RFC and PCNA to load the clamp on the primed site as well one of the DNA polymerases (Pol ɛ; or δ). Length of DNA at each time point was assessed by laser scans of the data in Panels A and B. The graphs illustrated in panel A and B are ImageQuant analysis scans (arbitrary units) of 1’ and 2’ (panel a – for Pol ɛ) and 20” and 40” (panel b for Pol δ) of replication reaction products. Scans were analyzed by fitting to a single Gaussian distribution (black lines) and the numbers shown (together with the standard errors of parameters) in either blue or red correspond to the usage of eSSB or yRPA proteins during the replication reactions. Panels (c) and (d): Rates of Pol ɛ and Pol δ extension using either E. coli SSB (blue) or RPA (red).
Supplementary Figure 5 Pol δ takes over PCNA-primed ssDNA from a moving Pol ɛ.
The reaction was performed using PCNA-primed φX174 ssDNA as described in the Extended Experimental Procedures with the following exceptions: 100 nM Pol ɛ was included in the preincubation, and 5 nM Pol δ was added 10 s after DNA synthesis was initiated by adding dNTPs. Time points reflect the time after addition of Pol δ.
Supplementary Figure 6 Purified yeast Pol δ and Pol ɛ.
Pol δ and Pol ɛ were purified as described in Extended Experimental Procedures. 5 μg of the purified protein complexes were separated on a 8 % SDS-PAGE gel and stained with Commassie Brilliant Blue. The positions of the four Pol ɛ, and three Pol δ subunits are indicated to the right of their respective bands in the gel.
Supplementary Figure 7 Uncropped gels for figures in main manuscript.
Uncropped gels of (A) Fig. 2, (B) Fig. 4 and (C) Fig. 5, panel B of the main manuscript. For the gels in Figures 2 and 4 we cropped out the Molecular Weight Markers since their intensities were overpowering the signal of the experimental reactions. The gel was cropped in Figure 5 due to losses that resulted in loading un-equivalent reaction product at the 10 min time points of Pol ɛ/SSB. However, the 1,2,5 min time points revealed the full time course to final product in these reactions.
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Georgescu, R., Langston, L., Yao, N. et al. Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork. Nat Struct Mol Biol 21, 664–670 (2014). https://doi.org/10.1038/nsmb.2851
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DOI: https://doi.org/10.1038/nsmb.2851
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