Okazaki fragment processing:: Modulation of the strand displacement activity of DNA polymerase δ by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1
Okazaki fragment processing:: Modulation of the strand displacement activity of DNA polymerase δ by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1
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DOI：
10.1073/pnas.251193198
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发表时间：
2001-12-04
影响因子：
11.1
通讯作者：
Hübscher, U
中科院分区：
文献类型：
--
作者：
Maga, G;Villani, G;Hübscher, U
在真核生物的染色体复制过程中，DNA聚合酶Delta对DNA合成的先导链和滞后链都是必不可少的。POL Delta参与了Okazaki片段的成熟过程，用于新合成片段的延伸，以及先前存在的下游片段的RNA/DNA片段的位移，产生了作为DNA2和Flat核酸内切酶-1(Fen 1)核酸内切酶靶标的中间折叠结构。使用单链微环模板和退火的RNA/DNA引物，我们可以通过与DNA合成耦合的polDelta来测量链移位。我们的结果表明，仅polDelta就可以通过双链DNA区域以分布的方式合成多达72个核苷酸。增殖细胞核抗原(增殖细胞核抗原)降低了polDelta的模板解离率，从而增加了合成和链置换的过程，而复制蛋白A(RP-A)通过产生一个锁定的片断DNA结构将移位片段的大小限制在20-30个核苷酸，该结构是将Fen 1移位片段加工成可连接产物的底物。我们的数据支持Okazaki片段处理模型，其中DNA聚合酶Delta的链置换活性受增殖细胞核抗原、RP-A和Fen 1的协同作用调节。
DNA polymerase (pol) delta is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol delta has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA segment of the preexisting downstream fragment generating an intermediate flap structure that is the target for the Dna2 and flap endonuclease-1 (Fen 1) endonucleases. Using a single-stranded minicircular template with an annealed RNA/DNA primer, we could measure strand displacement by pol delta coupled to DNA synthesis. Our results suggested that pol delta alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol delta, thus increasing the processivity of both synthesis and strand displacement, whereas replication protein A (RP-A) limited the size of the displaced fragment down to 20-30 nucleotides, by generating a "locked" flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase delta is modulated by the concerted action of PCNA, RP-A and Fen 1.