Dna2 Exhibits a Unique Strand End-dependent Helicase Function
Dna2 Exhibits a Unique Strand End-dependent Helicase Function
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DOI：
10.1074/jbc.m110.165191
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发表时间：
2010-12-01
影响因子：
4.8
通讯作者：
Bambara, Robert A.
中科院分区：
文献类型：
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作者：
Balakrishnan, Lata;Polaczek, Piotr;Bambara, Robert A.
Dna2 核酸内切酶/解旋酶参与真核 DNA 处理，包括冈崎片段处理过程中产生的长瓣的切割。其不寻常的底物相互作用包括识别和结合瓣基底，然后穿过瓣的 5' 端，并定期裂解以产生长度类似于 5 nt 的最终产物。封闭 5' 端可防止裂解。 Dna2 ATP 驱动的 5' 至 3' DNA 解旋酶功能促进 Dna2 在皮瓣上的运动，可能有助于其核酸酶功能。在这里，我们演示了使用两种不同的核酸酶死亡 Dna2 突变体，在模拟冈崎片段的底物上，Dna2 必须穿到未封闭的 5' 瓣上才能显示解旋酶活性。这一要求在具有数千个核苷酸长度的单链区域的底物上得以维持。据我们所知，这是对真核解旋酶的首次描述，该解旋酶无法在内部加载到其跟踪链上，而是必须从末端进入。从生物学角度来看，加载要求可能有助于解旋酶与 Dna2 核酸酶功能协调，以防止在 DNA 处理过程中产生不期望的长瓣。
Dna2 endonuclease/helicase participates in eukaryotic DNA transactions including cleavage of long flaps generated during Okazaki fragment processing. Its unusual substrate interaction consists of recognition and binding of the flap base, then threading over the 5'-end of the flap, and cleaving periodically to produce a terminal product similar to 5 nt in length. Blocking the 5'-end prevents cleavage. The Dna2 ATP-driven 5' to 3' DNA helicase function promotes motion of Dna2 on the flap, presumably aiding its nuclease function. Here we demonstrate using two different nuclease-dead Dna2 mutants that on substrates simulating Okazaki fragments, Dna2 must thread onto an unblocked 5' flap to display helicase activity. This requirement is maintained on substrates with single-stranded regions thousands of nucleotides in length. To our knowledge this is the first description of a eukaryotic helicase that cannot load onto its tracking strand internally but instead must enter from the end. Biologically, the loading requirement likely helps the helicase to coordinate with the Dna2 nuclease function to prevent creation of undesirably long flaps during DNA transactions.