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. 2009 Dec 17;462(7275):940-3.
doi: 10.1038/nature08611. Epub 2009 Nov 18.

Coordinating DNA replication by means of priming loop and differential synthesis rate

Manjula Pandey  1 , Salman Syed, Ilker Donmez, Gayatri Patel, Taekjip Ha, Smita S Patel
Affiliations

Coordinating DNA replication by means of priming loop and differential synthesis rate

Manjula Pandey et al. Nature. .

Abstract

Genomic DNA is replicated by two DNA polymerase molecules, one of which works in close association with the helicase to copy the leading-strand template in a continuous manner while the second copies the already unwound lagging-strand template in a discontinuous manner through the synthesis of Okazaki fragments. Considering that the lagging-strand polymerase has to recycle after the completion of every Okazaki fragment through the slow steps of primer synthesis and hand-off to the polymerase, it is not understood how the two strands are synthesized with the same net rate. Here we show, using the T7 replication proteins, that RNA primers are made 'on the fly' during ongoing DNA synthesis and that the leading-strand T7 replisome does not pause during primer synthesis, contrary to previous reports. Instead, the leading-strand polymerase remains limited by the speed of the helicase; it therefore synthesizes DNA more slowly than the lagging-strand polymerase. We show that the primase-helicase T7 gp4 maintains contact with the priming sequence during ongoing DNA synthesis; the nascent lagging-strand template therefore organizes into a priming loop that keeps the primer in physical proximity to the replication complex. Our findings provide three synergistic mechanisms of coordination: first, primers are made concomitantly with DNA synthesis; second, the priming loop ensures efficient primer use and hand-off to the polymerase; and third, the lagging-strand polymerase copies DNA faster, which allows it to keep up with leading-strand DNA synthesis overall.

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Figures

Figure 1
Figure 1. Primer synthesis occurs concomitant with DNA unwinding and synthesis
a, Sequencing gel shows RNA primers (left) made by T7 replisome (600 nM) on ts40P-50% (300 nM) with dNTPs (1 mM each), CTP (0.5 mM), α32P-CTP, ATP (1 mM), MgCl2 (4 mM free), and dT90 trap (3 µM) at 18 °C in replication buffer. Total yield of RNA primers is shown on the right panel. b, Representative Cy3 and Cy5 intensity traces showing DNA unwinding by single-molecule FRET with T7 replisome (50 nM), 1 mM each dNTPs, ATP, and CTP, and 4 mM free Mg2+ (23±1 °C). Additional traces are shown in Supplementary Fig. 4. The FRET time course and dwell time histograms (right) determinations are described in the Methods. Priming and control substrates show high FRET before unwinding and similar FRET decrease time Δt due to unwinding/synthesis. c, Polyacrylamide sequencing gels show progressive elongation of fluorescein labelled DNA primer by T7 replisome on ts40–50% (left) and ts40P-50% (right) under the same conditions as in (a). The nucleotide incorporation rates and error bars (right panels) were calculated from the global fit to the polymerization model (Supplementary Information), and the average DNA primer elongation rates are shown with the standard error of the mean.
Figure 2
Figure 2. Priming loop: Primase domain maintains contact with the priming-sequence during replication
a, The cartoon shows that while the primase domain remains bound to the priming sequence to make RNA (red), nascent lagging-strand template forms a priming loop. b, Fluorescently labeled DNA fork to investigate priming loop. c, Top: Cy3 (donor, green) and Cy5 (acceptor, red) intensity time traces during DNA synthesis by T7 replisome. (i) - (iv) labels correspond to the various DNA states in the cartoons on the left. Middle: FRET efficiency vs. time. Bottom: Representative Cy3 and Cy5 intensity traces on the control fork without the priming sequence. d, Cumulative fraction vs. the indicated time intervals determined from single molecule time traces. The time intervals measured are marked by the arrows in c. 40 molecules from one experiment were used to build the curves (see Methods). Supplementary Fig 10 shows additional representative time traces.
Figure 3
Figure 3. Lagging-strand synthesis is faster than leading-strand synthesis
a, Sequencing gels showing the progressive elongation of fluorescein labeled DNA primer by T7 replisome (200 nM) on 100 nM of ts40 (left) and p/t40 (right) with T7 gp2.5 (5 µM) under the same conditions as in Fig. 1a, without dT90 trap. b, The rate constants of individual nucleotide addition steps. Errors were calculated from the global fits to the polymerization model. Standard error of mean are reported for the average DNA primer elongation rate constant.
Figure 4
Figure 4. Model of T7 DNA replication
The leading-strand template (yellow) is copied continuously by the cooperative action of T7 DNA polymerase and T7 gp4 while the lagging-strand template (in blue coated with gp2.5) is synthesized by T7 DNA polymerase through Okazaki fragment synthesis. The physical coupling of the leading and lagging-strand polymerases via T7 gp4 and gp2.5 interactions creates a Trombone loop. The priming loop (coated with gp2.5) is created between the physically linked primase and helicase domains of T7 gp4 due to ongoing DNA synthesis during primer synthesis. The priming loop keeps the nascent primer within physical reach of the lagging-strand polymerase. Upon primer hand-off to the lagging-strand polymerase, the priming loop becomes part of the Trombone loop.
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