Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication.
Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication.
复制标题
猿猴病毒 40 T 抗原解旋酶亲水通道在 DNA 复制中的作用。
DOI：
10.1128/jvi.00003-07
复制
发表时间：
2007
影响因子：
5.4
通讯作者：
Simmons,DanielT
中科院分区：
文献类型：
--
作者：
Wang,Weiping;Manna,David;Simmons,DanielT
猿猴病毒40（SV 40）六聚体解旋酶由位于每个六聚体内的相邻大层结构域之间的中央通道和六个亲水通道组成。为了研究亲水通道在SV 40 DNA复制中的功能，在不直接参与蛋白质-蛋白质接触的位点引入了一系列单点取代。突变体的特点是生化在各种方式。所有突变体在没有DNA的情况下正常寡聚化。有趣的是，10个突变体中有8个不能解绕含起始DNA片段，其中9个完全不能支持SV 40 DNA在体外复制。变异体根据生化特性分为四类。A类突变体结合DNA正常，ATP酶和解旋酶活性正常，但不能解旋起始DNA和支持SV 40 DNA复制。在低蛋白浓度下，B类突变体在单链DNA和起始DNA结合方面受到损害。它们在解旋酶活性和起始解旋以及支持DNA复制方面有缺陷。C类和D类突变体在低蛋白浓度下具有高于正常的单链DNA结合活性。C类突变体不能分离起始DNA并支持DNA复制。D类突变体正常解绕起源DNA，但在其支持DNA复制的能力方面受到损害。总之，这些结果表明，亲水性通道在SV 40 DNA从原点解旋和解旋酶内所得单链的放置中具有积极作用。
The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.