Processing of G4 DNA by Dna2 helicase/nuclease and replication protein a (RPA) provides insights into the mechanism of Dna2/RPA substrate recognition
Processing of G4 DNA by Dna2 helicase/nuclease and replication protein a (RPA) provides insights into the mechanism of Dna2/RPA substrate recognition
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DOI：
10.1074/jbc.m802244200
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发表时间：
2008-09-05
影响因子：
4.8
通讯作者：
Campbell, Judith L.
中科院分区：
文献类型：
--
作者：
Masuda-Sasa, Taro;Polaczek, Piotr;Campbell, Judith L.
通常在端粒和rDNA阵列中发现的富含多鸟嘌呤的DNA序列已被证明可以组装成称为G四联体或G4 DNA的结构，该结构由碱基堆叠的G四联体（一种由四个氢键鸟嘌呤组成的排列）来稳定。G4 DNA结构对DNA复制和修复过程中产生的许多解旋酶和核酸酶具有抗性。后链DNA复制蛋白Dna2在端粒中具有独特的定位，并在端粒新生生物发生中发挥作用，这促使我们研究了Dna2在G4含DNA底物上的活性。我们发现，酵母Dna2与由酵母端粒重复序列形成的G4 DNA结合的亲和力比与相同序列的单链DNA结合的亲和力高25倍。人类Dna2也结合G4 dna。酵母和人类dna的解旋酶活性都能有效解旋G4 dna。另一方面，酵母和人类DNA 2的核酸酶活性都因G4 DNA的形成而减弱，其抑制程度取决于G4结构的拓扑结构。这种抑制作用可以通过复制蛋白A来克服。复制蛋白A可以刺激Dna2的5'- 3'-核酸酶活性；然而，我们继续证明这种蛋白质抑制Dna2的3'-至5'-外显子/内切酶活性。这些观察结果讨论了Dna2在解决冈崎片段加工和端粒延长过程中出现的G4二级结构中的可能作用。
The polyguanine-rich DNA sequences commonly found at telomeres and in rDNA arrays have been shown to assemble into structures known as G quadruplexes, or G4 DNA, stabilized by base-stacked G quartets, an arrangement of four hydrogen-bonded guanines. G4 DNA structures are resistant to the many helicases and nucleases that process intermediates arising in the course of DNA replication and repair. The lagging strand DNA replication protein, Dna2, has demonstrated a unique localization to telomeres and a role in de novo telomere biogenesis, prompting us to study the activities of Dna2 on G4 DNA-containing substrates. We find that yeast Dna2 binds with 25-fold higher affinity to G4 DNA formed from yeast telomere repeats than to single-stranded DNA of the same sequence. Human Dna2 also binds G4 DNAs. The helicase activities of both yeast and human Dna2 are effective in unwinding G4 DNAs. On the other hand, the nuclease activities of both yeast and human Dna2 are attenuated by the formation of G4 DNA, with the extent of inhibition depending on the topology of the G4 structure. This inhibition can be overcome by replication protein A. Replication protein A is known to stimulate the 5'- to 3'-nuclease activity of Dna2; however, we go on to show that this same protein inhibits the 3'- to 5'-exo/endonuclease activity of Dna2. These observations are discussed in terms of possible roles for Dna2 in resolving G4 secondary structures that arise during Okazaki fragment processing and telomere lengthening.