Coupling of DNA helicase and endonuclease activities of yeast Dna2 facilitates Okazaki fragment processing.
Coupling of DNA helicase and endonuclease activities of yeast Dna2 facilitates Okazaki fragment processing.
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DOI：
10.1074/jbc.m111026200
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发表时间：
2002-07-19
影响因子：
4.8
通讯作者：
Seo, YS
中科院分区：
文献类型：
--
作者：
Bae, SH;Kim, DW;Seo, YS
酿酒酵母 Dna2 具有解旋酶和核酸内切酶活性。其核酸内切酶活性至关重要，非常适合去除冈崎片段的 RNA-DNA 引物。相反，其解旋酶活性虽然是最佳生长所必需的，但当细胞生长速率降低时则不再是必需的。这些发现表明 Dna2 的 DNA 解旋活性在冈崎片段加工中发挥辅助作用。为了解决这个问题，我们使用两种突变蛋白 Dna2D657A 和 Dna2K1080E（分别仅包含解旋酶或核酸内切酶活性）检查了 Dna2 解旋酶活性是否影响其内在核酸内切酶活性。使用 Dna2D657A 和 Dna2K1080E 酶的混合物进行的实验表明，当酶沿着底物易位时，Dna2 的核酸内切酶活性会发生单链 DNA 的切割。此外，DNA解旋活性有效地去除了瓣结构中形成的二级结构，这进一步得到了复制蛋白A的帮助。我们的结果表明，Dna2解旋活性通过其内在的核酸内切酶活性在促进瓣DNA去除中发挥了作用。
Saccharomyces cerevisiae Dna2 possesses both helicase and endonuclease activities. Its endonuclease activity is essential and well suited to remove RNA-DNA primers of Okazaki fragments. In contrast, its helicase activity, although required for optimal growth, is not essential when the rate of cell growth is reduced. These findings suggest that DNA unwinding activity of Dna2 plays an auxiliary role in Okazaki fragment processing. To address this issue, we examined whether the Dna2 helicase activity influenced its intrinsic endonuclease activity using two mutant proteins, Dna2D657A and Dna2K1080E, which contain only helicase or endonuclease activity, respectively. Experiments performed with a mixture of Dna2D657A and Dna2K1080E enzymes revealed that cleavage of a single-stranded DNA by endonuclease activity of Dna2 occurs while the enzyme translocates along the substrate. In addition, DNA unwinding activity efficiently removed the secondary structure formed in the flap structure, which was further aided by replication protein A. Our results suggest that the Dna2 unwinding activity plays a role in facilitating the removal of the flap DNA by its intrinsic endonuclease activity.