Nonspecific double-stranded DNA binding activity of simian virus 40 large T antigen is involved in melting and unwinding of the origin.
Nonspecific double-stranded DNA binding activity of simian virus 40 large T antigen is involved in melting and unwinding of the origin.
复制标题
猿猴病毒40大T抗原的非特异性双链DNA结合活性参与起源的解链和解旋。
DOI：
10.1128/jvi.77.23.12720-12728.2003
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发表时间：
2003
影响因子：
5.4
通讯作者：
Simmons,DanielT
中科院分区：
文献类型：
--
作者：
Jiao,Junfang;Simmons,DanielT
解旋酶活性是T抗原解开猿猴病毒40起源所必需的。我们先前将该活性定位于残基131和616。在这项研究中，我们产生了一系列的解旋酶结构域中的单点取代的突变体，以发现解旋酶功能所需的其他潜在活动。产生了许多DNA解旋缺陷突变体。其中4个突变体（456 RA、460 ED、462 GA和499 DA）水解ATP的能力正常，并且能够在原始DNA存在下结合成双六聚体。此外，它们具有正常的结合单链DNA的能力。然而，它们在解旋含有起始的DNA片段和与M13部分双链体DNA底物进行解旋酶反应中严重受损。有趣的是，这些突变体保留了一些与人工复制叉进行解旋酶反应的能力，表明它们的内在解旋酶活性是功能性的。有趣的是，这些突变体几乎完全失去了非特异性结合双链DNA的能力。这些突变体也未能融化起源的早期回文区。综上所述，这些结果表明，突变破坏了一种新的活性所需的解旋的起源。这种活性依赖于非特异性结合DNA的能力，在其缺失的情况下，T抗原不能在结构上扭曲并随后解开起源。
Helicase activity is required for T antigen to unwind the simian virus 40 origin. We previously mapped this activity to residues 131 and 616. In this study, we generated a series of mutants with single-point substitutions in the helicase domain to discover other potential activities required for helicase function. A number of DNA unwinding-defective mutants were generated. Four of these mutants (456RA, 460ED, 462GA, and 499DA) were normal in their ability to hydrolyze ATP and were capable of associating into double hexamers in the presence of origin DNA. Furthermore, they possessed normal ability to bind to single-stranded DNA. However, they were severely impaired in unwinding origin-containing DNA fragments and in carrying out a helicase reaction with an M13 partial duplex DNA substrate. Interestingly, these mutants retained some ability to perform a helicase reaction with artificial replication forks, indicating that their intrinsic helicase activity was functional. Intriguingly, these mutants had almost completely lost their ability to bind to double-stranded DNA nonspecifically. The mutants also failed to melt the early palindrome region of the origin. Taken together, these results indicate that the mutations have destroyed a novel activity required for unwinding of the origin. This activity depends on the ability to bind to DNA nonspecifically, and in its absence, T antigen is unable to structurally distort and subsequently unwind the origin.