Langston Lance D, O'Donnell Mike
Howard Hughes Medical Institute, Rockefeller University, New York, New York 10065, USA.
J Biol Chem. 2008 Oct 24;283(43):29522-31. doi: 10.1074/jbc.M804488200. Epub 2008 Jul 16.
In most cells, 100-1000 Okazaki fragments are produced for each replicative DNA polymerase present in the cell. For fast-growing cells, this necessitates rapid recycling of DNA polymerase on the lagging strand. Bacteria produce long Okazaki fragments (1-2 kb) and utilize a highly processive DNA polymerase III (pol III), which is held to DNA by a circular sliding clamp. In contrast, Okazaki fragments in eukaryotes are quite short, 100-250 bp, and thus the eukaryotic lagging strand polymerase does not require a high degree of processivity. The lagging strand polymerase in eukaryotes, polymerase delta (pol delta), functions with the proliferating cell nuclear antigen (PCNA) sliding clamp. In this report, Saccharomyces cerevisiae pol delta is examined on model substrates to gain insight into the mechanism of lagging strand replication in eukaryotes. Surprisingly, we find pol delta is highly processive with PCNA, over at least 5 kb, on Replication Protein A (RPA)-coated primed single strand DNA. The high processivity of pol delta observed in this report contrasts with its role in synthesis of short lagging strand fragments, which require it to rapidly dissociate from DNA at the end of each Okazaki fragment. We find that this dilemma is solved by a "collision release" process in which pol delta ejects from PCNA upon extending a DNA template to completion and running into the downstream duplex. The released pol delta transfers to a new primed site, provided the new site contains a PCNA clamp. Additional results indicate that the collision release mechanism is intrinsic to the pol3/pol31 subunits of the pol delta heterotrimer.
在大多数细胞中,每个细胞内存在的复制性DNA聚合酶会产生100 - 1000个冈崎片段。对于快速生长的细胞而言,这就需要滞后链上的DNA聚合酶快速循环利用。细菌产生长的冈崎片段(1 - 2 kb),并利用高度持续合成的DNA聚合酶III(pol III),它通过一个环形滑动夹与DNA相连。相比之下,真核生物中的冈崎片段相当短,为100 - 250 bp,因此真核生物的滞后链聚合酶不需要高度的持续合成能力。真核生物中的滞后链聚合酶,即聚合酶δ(pol δ),与增殖细胞核抗原(PCNA)滑动夹共同发挥作用。在本报告中,对酿酒酵母pol δ在模型底物上进行了研究,以深入了解真核生物滞后链复制的机制。令人惊讶的是,我们发现pol δ与PCNA一起在复制蛋白A(RPA)包被的引发单链DNA上具有至少超过5 kb的高度持续合成能力。本报告中观察到的pol δ的高持续合成能力与其在短滞后链片段合成中的作用形成对比,在短滞后链片段合成中,它需要在每个冈崎片段末端迅速从DNA上解离。我们发现这个难题通过一种“碰撞释放”过程得以解决,在这个过程中,pol δ在将DNA模板延伸至完成并遇到下游双链体时从PCNA上弹出。释放的pol δ转移到一个新的引发位点,前提是新位点含有一个PCNA夹。其他结果表明,碰撞释放机制是pol δ异源三聚体的pol3/pol31亚基所固有的。