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

Abstract

Genomic DNA is replicated by two DNA polymerase molecules: one 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 synthesis of Okazaki fragments 1, 2 . Considering the lagging-strand polymerase has to recycle after every Okazaki fragment through the slow steps of primer synthesis and hand-off to the polymerase 3-5 , it is not understood how the two strands are synthesized with the same net rate [6][7][8][9] . Here, we show with the T7 replication proteins 10, 11 that RNA primers are made on the fly during ongoing DNA synthesis, and the leading-strand T7 replisome does not pause during primer synthesis contrary to previous reports 12,13 . Instead, the leading-strand polymerase remains limited by the speed of the helicase 14 , so synthesizes DNA at a slower rate than the lagging-strand polymerase. We show that the primase-helicase T7 gp4 maintains contact with the priming sequence during ongoing DNA synthesis; hence, the nascent lagging-strand template organizes into a priming loop that keeps the primer in physical proximity to the replication complex. Our findings provides three synergistic mechanisms of coordination: 1) Primers are made concomitantly with DNA synthesis; 2) the priming-loop assures efficient primer utilization and hand-off to the polymerase; 3) the laggingstrand polymerase copies DNA faster, which allows it to keep up with leading-strand DNA synthesis overall.To investigate the functional cooperativity between the enzymatic activities of the T7 replication complex, we measured the kinetics of DNA unwinding, DNA synthesis, and primer synthesis on synthetic replication fork substrates with and without the T7 primingsequence (3'-CTGGG, Supplementary Table 1). Efficient synthesis of RNA primers from 2mer to 5mer by T7 replisome (T7 gp4 and T7 DNA polymerase) was observed on the priming fork ( Fig. 1a and Supplementary Fig. 1) with half-life of ~0.5 s and yield >60% (Fig. 1a, right). T7 gp4 alone also makes RNA primers on this priming fork, but at ~10-fold * Correspondence: patelss@umdnj.edu and tjha@illinois.edu.Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Contributions M.P. purified T7 gp5, T7 gp4 and constructed DNA substrates for the priming loop studies, and obtained and analyzed 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 analyzed 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, analyzed the data, and wrote the manuscript. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 December 17. Supplementary Fig. 1), consistent with polymerase assistance of the helicase rate 14 . An average 46% yield of primer synthesis with fork...

Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

“…In the second model, coordination is achieved by different synthesis rates of the leading and the lagging strands. Contrary to the results described by Lee et al (32), the studies of the T7 replisome by Pandey et al (37) indicated that lagging-strand DNA synthesis is 38% faster than leading-strand DNA synthesis. In addition, no pausing of replisome movement was detected during primer synthesis.…”

Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome

“…7B). Gp4, translocating in a 5′-3′ direction, encounters a PRS and synthesizes a primer, pausing the replisome while the primer remains bound to gp4 (33)(34)(35) before its transfer to DNA polymerase. In this step, gp2.5 promotes more efficient full-length primer synthesis.…”

Single-Molecule Studies of the Replisome

“…Recent single-molecule experiments have demonstrated the existence of an alternative mechanism to couple primase activity to leading-strand synthesis (72,82). The formation of a small, ssDNA loop between helicase and primase would allow helicase-mediated unwinding to continue while the primase remains bound to the lagging strand to synthesize a primer.…”

“…It is not well understood how these slow enzymatic steps can take place at the lagging strand without losing coordination with the continuous and fast leadingstrand synthesis (62,93,108). Does the laggingstrand DNA polymerase synthesize faster than the leading-strand DNA polymerase to compensate for the time lost during polymerase cycling (14,82)? Or does leading-strand synthesis transiently halt to prevent it from outpacing lagging-strand synthesis (64)?…”