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Resolving individual steps of Okazaki-fragment maturation at a millisecond timescale

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Abstract

DNA polymerase delta (Pol δ) is responsible for elongation and maturation of Okazaki fragments. Pol δ and the flap endonuclease FEN1, coordinated by the PCNA clamp, remove RNA primers and produce ligatable nicks. We studied this process in the Saccharomyces cerevisiae machinery at millisecond resolution. During elongation, PCNA increased the Pol δ catalytic rate by >30-fold. When Pol δ invaded double-stranded RNA–DNA representing unmatured Okazaki fragments, the incorporation rate of each nucleotide decreased successively to 10–20% that of the preceding nucleotide. Thus, the nascent flap acts as a progressive molecular brake on the polymerase, and consequently FEN1 cuts predominantly single-nucleotide flaps. Kinetic and enzyme-trapping experiments support a model in which a stable PCNA–DNA–Pol δ–FEN1 complex moves processively through iterative steps of nick translation, ultimately completely removing primer RNA. Finally, whereas elongation rates are under dynamic dNTP control, maturation rates are buffered against changes in dNTP concentrations.

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Figure 1: PCNA stimulates the catalytic rate of Pol δ.
Figure 2: Strand-displacement synthesis by PCNA–Pol δ.
Figure 3: Strand-displacement synthesis and idling by wild-type Pol δ.
Figure 4: Nick translation by Pol δ and FEN1.
Figure 5: Processivity of the nick-translation machinery.
Figure 6: Model for short-flap maintenance and nick translation.

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  • 19 April 2016

    In the version of this article initially published online, there were partial omissions within the schematics depicted in Figure 5a,b. These errors have been corrected for the print, HTML and PDF versions of the article.

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Acknowledgements

The authors thank J. Majors, R. Galletto, and T. Lohman for critical discussions during the progress of this work, and C. Stith for protein purification. This work was supported in part by the US National Institutes of Health (GM032431 to P.B.) and from the US-Israel Binational Science Foundation (2013358 to P.B.).

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J.L.S. and P.M.B. designed experiments and analyzed data. J.L.S. performed all experiments. J.L.S. and P.M.B. wrote the manuscript.

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Correspondence to Peter M Burgers.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Analysis of replication rates by Pol d.

(a) Electrophoretic Mobility Shift Assay (EMSA) of Pol δ-DV binding to template-primer DNA (Fried, M. & Crothers, D.M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505-6518 (1981)). 20 nM DNA was incubated with increasing concentrations of Pol δ-DV. Complexes were resolved on a 5%, 1X TBE native polyacrylamide gel. Analysis was carried out with 20 nM template as this was the pre-incubation concentration of DNA prior to mixing with an equal volume of dNTP solution in the rapid-quench apparatus. (b) Single nucleotide incorporation by Pol δ-DV alone (no PCNA), identical to described in Fig. 1 b,c, with either 250 μM or 500 μM dTTP. Time courses were fit to single exponentials, representative of first-order kinetics. (c) Quantification of replication time courses of a homopolymeric DNA by PCNA-Pol d. Experiments were performed identically to that in Fig. 1d, but with either 250 μM or 500 μM each of dTTP and dATP. Median analysis is described in detail in “Supplementary Experimental Procedures”. (d) Replication through a homopolymeric stretch of DNA by PCNA-Pol δ; images of gels quantified in Fig. 1e. DNA template was pre-incubated with subsets of an enzyme mix containing Pol δ-DV, RPA, PCNA, RFC, and AMP-CPP. Omissions from this standard reaction mix are noted. Reactions were initiated with 250 μM dTTP and dATP each to allow extension of the 29-mer primer to a 50-mer product. (e) Effect of RPA on Pol δ-DV extension in the absence of PCNA. Primer extension reactions were performed on substrate described in Fig. 1a, with and without 50 nM RPA pre-bound to the single-stranded DNA template. Reactions were initiated with either 250 μM dATP or 250 μM each dATP and dTTP as noted. (f) Primer extension reactions by PCNA-Pol δ. Standard replication reactions on the template shown in Fig. 1a containing all components were initiated with either 250 μM each of all four dNTPs, all four dNTPs at S. cerevisiae physiological concentrations (16 μM dATP, 14 μM dCTP, 12 μM dGTP, 30 μM dTTP), or all four dNTPs and rNTPs at S. cerevisiae physiological concentrations (dNTPs as before plus 3 mM ATP, 0.5 mM CTP, 0.7 mM GTP, 1.7 mM UTP). (g) Quantification of data from f. The median extension product at each time point was determined and plotted as a function of time. Each curve was fit to a single exponential.

Supplementary Figure 2 Strand-displacement synthesis by exonuclease-deficient Pol d.

(a, b) Global kinetic modeling of data from Fig. 2a to the respective kinetic models shown. KinTek Explorer software was used to perform fits (Johnson, K.A., Simpson, Z.B. & Blom, T. Global kinetic explorer: a new computer program for dynamic simulation and fitting of kinetic data. Anal Biochem 387, 20-9 (2009)) The fitting shown in Fig. 2b was performed in a model-free manner, providing information concerning the observed macroscopic rates of strand displacement synthesis, but not about the molecular mechanism of the observed slow-down. In an attempt to better define this molecular mechanism, we performed global kinetic fitting of the data in Fig. 2a to two different models. First, we globally fit the data to the simplest model, in which the flap inhibits the actual rate of extension by Pol δ and a longer flap inhibits more effectively in (a). This model yielded a poor fit, especially for the +2 and +3-nt displacement products. The rates obtained from this global fit were comparable to those generated by fitting each product curve to the sum of two exponentials individually (compare (a) with Fig. 2b). A second, more complex mechanism was considered, in which Pol δ equilibrates between two states during strand displacement, one that is competent for further extension and one incompetent for extension (b). Fitting to such a model provided a better fit to the data, as is expected by the inclusion of more variables. Our modeling indicates that the incompetent state is not significantly populated during polymerization of single-strand DNA templates, but becomes increasingly more populated as strand displacement synthesis progresses. While several rates were not well defined by this model, we believe that its principle has merit because it provides a mechanistic explanation for Pol δ carrying out activities on flap substrates other than polymerization, such as idling and hand-off to FEN1. (c, d) Strand displacement time courses performed on the indicated Substrates I and II with either RNA or DNA-initiating blocking oligonucleotides. Select time points from these gels are shown in Fig. 2b. (e) Strand displacement time course performed on DNA Substrate III-RNA block. The reaction was performed identically to those in (c) and (d). (f) Quantitation of data from (e); Displacement products (0) and (+1) were fit to two exponentials, and (+2) and (≥+3) to single exponentials. (g) Global KinTek modeling using the simple model in (a). The experiments in (e-g) were carried out to show that the progressive slowdown observed during strand displacement synthesis in Substrates I and II was not the result of the specific DNA or RNA sequence used, but a consequence of the increasing length of the flap. In Substrate III, the dinucleotide stability for each pair of nucleotides within the four, 5’-proximal nucleotides was constant (5’-rGrGrGrC), yet the strand displacement time-course shows that rate of strand displacement synthesis progressively decreases as the nascent flap grows longer.

Supplementary Figure 3 Strand-displacement synthesis and idling by wild-type Pol d.

(a,c) Strand displacement time courses performed with Pol δ-wt as described in Fig. 3a. The substrate and enzymes were pre-incubated in the presence of dCTP and dGTP to prevent polymerase degradation of the primer and blocking oligonucleotide. (b,d) Quantification of products in a,c. Fractional occupancy was determined and select products are plotted. The nick position product (0), +1 position past nick, and the +2 and greater position were plotted for both the RNA-initiating block (a,b) and the DNA block (c,d) of Substrate I. The c plot is also in Figure 3, but is shown again for easier comparison.

Supplementary Figure 4 Nick translation by Pol δ and FEN1.

(a) Experimental design of nick translation assay. (b) Quantification of data from Fig. 2d (no FEN1) and Fig. 4b, top panel (+FEN1), showing nick position and +1 displacement product. (c) FEN1-cut products from Substrate I-RNA with 5’-labeled blocking oligonucleotide. The fraction of each product size is shown. The total of cut products and substrate remaining equals 1. (d) The ratio of (2-nt + 3-nt)/1-nt product formed during each subsequent 0.2 sec interval was plotted against assay time. The plot shows that at the start the 1-nt product predominated (ratio =0.3), while after 2 sec, the larger products predominated (ratio =4.5). (e) Nick translation assay on Substrate III-RNA block, as diagramed in a. (f) Quantification of e (+FEN1) and Supplementary Fig. 2e (no FEN1). Nick position (0), and +1 and +2 displacement products were plotted. (g) FEN1-cut products from Substrate III-RNA with 5’-labeled blocking oligonucleotide. The fraction of each product size is shown. The total of cut products and substrate remaining equals 1. (h) Median extension analysis of nick translation assays performed at 250 μM each dNTP (blue), and at physiological dNTP levels (red, concentrations listed in legend to Supplementary Fig. 1f). Data for saturating dNTPs are the same as in Fig. 4e. Data collected with low dNTP levels shows a lag in nick translation at early time points, which we attribute to slower gap filling and formation of the +1 flap at the lower, physiological dNTPs. Comparison of the two slopes in the linear range indicates that iterative nick translation proceeds at approximately the same rate at physiological as at saturating nucleotide concentrations. (i) Quench-flow assay with FEN1 and various flap-containing DNAs. Reactions were initiated by mixing FEN1 with DNA template. Other than the nick-containing template (green), DNAs contained a single extrahelical 3’-nucleotide complementary to the template. Templates then contained either 0 (blue), 1 (black), or 2 (purple) extrahelical 5’-rU bases, not complementary to the template. All templates were labeled with a 5’-32P on the strand cut by FEN1. The fraction of flap cut is plotted. These assays were carried out without PCNA since it was not efficiently loaded on the flap substrates.

Supplementary Figure 5 Processivity of the nick-translation machinery.

(a) Similar to Fig. 5a; strand displacement time-course by PCNA-Pol δ on Substrate I-DNA block. Standard reaction conditions were used, initiated with either 250 μM of each dNTP (high) or physiological levels of each dNTP (phys, concentrations listed in legend to Supplementary Fig. 1f). 10 μg/ml heparin was used as trap for free Pol δ-DV in noted lanes. (b) Similar to Supplementary Fig. 5a, lanes 1-6 (250 µM each dNTP), except Substrate I-RNA block was used instead of a DNA-block. (c) Companion to Fig. 5b; nick translation assay with forced single turnover of 40 nM FEN1-p, containing mutations in the FEN1 PIP-motif. DNA template was Substrate I-RNA block. Reactions were initiated with 250 μM each dNTP with or without 6 μM oligonucleotide FEN1 trap to trap free FEN1-p. The data show that the trap completely blocked FEN1-p action, even when it was pre-bound to the DNA-PCNA- Pol δ complex, indicating that it is not stably associated with this complex. (d) Nick translation assay with forced single turnover of 40 nM FEN1, with wild-type Pol δ. Reactions were initiated with 250 μM each dNTP with or without 6 μM oligonucleotide FEN1 trap to trap free FEN1. The data show that FEN1 is able to remain associated with the PCNA-wild-type Pol δ complex throughout nick translation (e) Testing the efficiency of the FEN1 oligonucleotide trap; FEN1 and FEN1-p cutting of labeled substrate containing a stable flap. Labeled DNA contained a single nucleotide 3’-flap and a two-nucleotide 5’-flap (both non-complementary to template), with a 3’-Cy3 label on the strand cut by FEN1. Reactions were initiated by mixing the enzyme with DNA template. To test the effectiveness of the oligonucleotide trap, FEN1 and FEN1-p were pre-incubated with excess trap template prior to incubation with labeled template. The structure of the trap substrate was identical to that of the labeled substrate.

Supplementary Figure 6 Median analysis of replication rates.

This analysis method was used to generate the data presented in Fig. 1e, 4e, and Supplementary Fig. 1c,g, 4h. (a) Plot profiles of all products, except starting material, were produced using ImageQuant (GE Healthcare). These profiles plotted the intensity signal in the gel against an arbitrary y-coordinate. Following background subtraction, we determined the position on the y-coordinate at which the median of the total lane signal was. This was defined as the point along the lane coordinate in which 50% of the signal in which 50% of the signal lay above and below. (b) Next, for each gel, a standard curve was produced, fit to a quadratic function, in order to convert the arbitrary y-coordinate values to a value represented in nucleotides. (c) After determining the median product for many points throughout an entire time-course, the plots were assembled.

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Stodola, J., Burgers, P. Resolving individual steps of Okazaki-fragment maturation at a millisecond timescale. Nat Struct Mol Biol 23, 402–408 (2016). https://doi.org/10.1038/nsmb.3207

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