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

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 fragments1,2. 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 polymerase3,4,5, it is not understood how the two strands are synthesized with the same net rate6,7,8,9. Here we show, using the T7 replication proteins10,11, 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 reports12,13. Instead, the leading-strand polymerase remains limited by the speed of the helicase14; 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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Figure 1: Primer synthesis occurs concomitantly with DNA unwinding and synthesis.
Figure 2: Priming loop: the primase domain maintains contact with the priming sequence during replication.
Figure 3: Lagging-strand synthesis is faster than leading-strand synthesis.
Figure 4: Model of T7 DNA replication.

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Acknowledgements

We thank C. M. Drain for a critical reading of the paper, K. Picha and S. Patel for preparation of the minicircle DNA, and S. Arslan and K. S. Lee for help with single-molecule FRET data analysis. This work was supported by NIH grants GM55310 (S.S.P.) and GM065367 (T.H.) and NSF grants 0822613 and 0646550 (T.H.). T.H. is an investigator with the Howard Hughes Medical Institute.

Author Contributions M.P. purified T7 gp5 and T7 gp4, and constructed DNA substrates for the priming-loop studies, and obtained and analysed all the ensemble DNA synthesis and primer synthesis experiments. S.S. developed robust single-molecule assays for observing DNA unwinding and priming-loop formation, and obtained and analysed all single-molecule data. I.D. and G.P. performed the ensemble unwinding experiments. M.P., S.S., S.S.P. and T.H. designed the experiments, analysed the data and wrote the manuscript.

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Correspondence to Taekjip Ha or Smita S. Patel.

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Pandey, M., Syed, S., Donmez, I. et al. Coordinating DNA replication by means of priming loop and differential synthesis rate . Nature 462, 940–943 (2009). https://doi.org/10.1038/nature08611

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