RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes
RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes
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DOI：
10.1038/35086609
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发表时间：
2001-07-26
期刊：
影响因子：
64.8
通讯作者：
Seo, YS
中科院分区：
文献类型：
--
作者：
Bae, SH;Bae, KH;Seo, YS
对猴病毒40 DNA复制过程中滞后链成熟的大量研究表明，冈崎片段的起始RNA引物在冈崎片段连接之前被两种核酸酶RNase HI和Fen 1的联合作用去除（1-5）。尽管这两种酶在体外的作用已经很好地确立（6），但酵母中的遗传分析显示，RNase HI和/或Fen 1的无效突变体不是致命的（7-9），这表明去除RNA可能需要额外的酶活性。一种这样的酶是酿酒酵母Dna 2解旋酶（10-12）/内切核酸酶（12），其对于细胞活力是必需的（13，14）并且非常适合于去除冈崎片段的RNA引物（15）。此外，Dna 2与参与冈崎片段的延伸或成熟的几种蛋白质在遗传和物理上相互作用（10，16）。在这里，我们表明，核酸内切酶Dna 2和Fen 1的作用顺序，以促进完全删除的引物RNA。这些酶的顺序作用由单链DNA结合蛋白，复制蛋白A（RPA）控制。我们的研究结果表明，冈崎片段在真核生物中的处理显着不同，是更复杂的比，发生在原核生物。我们提出了一种新的生化机制，真核生物冈崎片段的成熟。
Extensive work on the maturation of lagging strands during the replication of simian virus 40 DNA suggests that the initiator RNA primers of Okazaki fragments are removed by the combined action of two nucleases, RNase HI and Fen1, before the Okazaki fragments join(1-5). Despite the well established in vitro roles of these two enzymes(6), genetic analyses in yeast revealed that null mutants of RNase HI and/or Fen1 are not lethal(7-9), suggesting that an additional enzymatic activity may be required for the removal of RNA. One such enzyme is the Saccharomyces cerevisiae Dna2 helicase(10-12)/endonuclease(12), which is essential for cell viability(13,14) and is well suited to removing RNA primers of Okazaki fragments(15). In addition, Dna2 interacts genetically and physically with several proteins involved in the elongation or maturation of Okazaki fragments(10,16). Here we show that the endonucleases Dna2 and Fen1 act sequentially to facilitate the complete removal of the primer RNA. The sequential action of these enzymes is governed by a single-stranded DNA-binding protein, replication protein-A (RPA). Our results demonstrate that the processing of Okazaki fragments in eukaryotes differs significantly from, and is more complicated than, that occurring in prokaryotes. We propose a novel biochemical mechanism for the maturation of eukaryotic Okazaki fragments.