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Insights into Okazaki Fragment Synthesis by the T4 Replisome

2013; Elsevier BV; Volume: 288; Issue: 29 Linguagem: Inglês

10.1074/jbc.m113.485961

1083-351X

Danqi Chen, Hongjun Yue, Michelle M. Spiering, Stephen J. Benkovic,

Bacteriophages and microbial interactions

In this study, we employed a circular replication substrate with a low priming site frequency (1 site/1.1 kb) to quantitatively examine the size distribution and formation pattern of Okazaki fragments. Replication reactions by the T4 replisome on this substrate yielded a patterned series of Okazaki fragments whose size distribution shifted through collision and signaling mechanisms as the gp44/62 clamp loader levels changed but was insensitive to changes in the gp43 polymerase concentration, as expected for a processive, recycled lagging-strand polymerase. In addition, we showed that only one gp45 clamp is continuously associated with the replisome and that no additional clamps accumulate on the DNA, providing further evidence that the clamp departs, whereas the polymerase is recycled upon completion of an Okazaki fragment synthesis cycle. We found no support for the participation of a third polymerase in Okazaki fragment synthesis.Background: The T4 replisome duplicates the lagging DNA strand through discontinuous Okazaki fragments.Results: A third polymerase is not involved in repetitive Okazaki fragment synthesis.Conclusion: The T4 replisome recycles the lagging-strand polymerase but looses the clamp in each Okazaki fragment cycle through a collision or signaling pathway.Significance: Elucidation of the behavior of polymerase, clamp and clamp loader for Okazaki fragment synthesis broadens our understanding of coordinated DNA replication. In this study, we employed a circular replication substrate with a low priming site frequency (1 site/1.1 kb) to quantitatively examine the size distribution and formation pattern of Okazaki fragments. Replication reactions by the T4 replisome on this substrate yielded a patterned series of Okazaki fragments whose size distribution shifted through collision and signaling mechanisms as the gp44/62 clamp loader levels changed but was insensitive to changes in the gp43 polymerase concentration, as expected for a processive, recycled lagging-strand polymerase. In addition, we showed that only one gp45 clamp is continuously associated with the replisome and that no additional clamps accumulate on the DNA, providing further evidence that the clamp departs, whereas the polymerase is recycled upon completion of an Okazaki fragment synthesis cycle. We found no support for the participation of a third polymerase in Okazaki fragment synthesis. Background: The T4 replisome duplicates the lagging DNA strand through discontinuous Okazaki fragments. Results: A third polymerase is not involved in repetitive Okazaki fragment synthesis. Conclusion: The T4 replisome recycles the lagging-strand polymerase but looses the clamp in each Okazaki fragment cycle through a collision or signaling pathway. Significance: Elucidation of the behavior of polymerase, clamp and clamp loader for Okazaki fragment synthesis broadens our understanding of coordinated DNA replication.