Cysteine 111 affects coupling of single-stranded DNA binding to ATP hydrolysis in the herpes simplex virus type-1 origin-binding protein.
Cysteine 111 affects coupling of single-stranded DNA binding to ATP hydrolysis in the herpes simplex virus type-1 origin-binding protein.
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半胱氨酸 111 影响单链 DNA 与 1 型单纯疱疹病毒起源结合蛋白中 ATP 水解的偶联。
DOI：
10.1074/jbc.275.4.2931
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Boehmer,PE
中科院分区：
文献类型：
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作者：
Sampson,DA;Arana,ME;Boehmer,PE
单纯疱疹病毒1型起源结合蛋白（UL 9蛋白）通过解旋起源启动病毒复制。它具有序列特异性DNA结合活性、单链DNA结合活性、DNA解旋酶活性和受单链DNA强烈刺激的ATP酶活性。我们已经研究了半胱氨酸作为DNA解旋酶的作用。DNA解旋酶和DNA依赖的ATP酶活性可被还原剂激活，巯基特异性试剂N-乙基马来酰亚胺特异性灭活。为了鉴定导致这种现象的半胱氨酸，将ATP结合位点附近的保守半胱氨酸（半胱氨酸111）突变为丙氨酸。UL 9 C111 A蛋白在其DNA解旋酶和依赖于DNA的ATP酶活性方面表现出缺陷，并且不能在体内支持来源特异性DNA复制。动力学分析表明，这些缺陷是由于单链DNA不能诱导UL 9 C111 A蛋白中的高亲和力ATP结合。UL 9 C111 A蛋白的DNA依赖性ATP酶活性对N-乙基马来酰亚胺具有抗性，而其DNA解旋酶活性保持敏感。因此，UL 9蛋白对N-乙基马来酰亚胺的敏感性是由于至少两个半胱氨酸。半胱氨酸111参与将单链DNA结合偶联至ATP结合和随后的水解，而第二个半胱氨酸参与将ATP水解偶联至DNA解旋。
Herpes simplex virus type-1 origin-binding protein (UL9 protein) initiates viral replication by unwinding the origins. It possesses sequence-specific DNA-binding activity, single-stranded DNA-binding activity, DNA helicase activity, and ATPase activity that is strongly stimulated by single-stranded DNA. We have examined the role of cysteines in its action as a DNA helicase. The DNA helicase and DNA-dependent ATPase activities of UL9 protein were stimulated by reducing agent and specifically inactivated by the sulfhydryl-specific reagentN-ethylmaleimide. To identify the cysteine responsible for this phenomenon, a conserved cysteine in the vicinity of the ATP-binding site (cysteine 111) was mutagenized to alanine. UL9C111A protein exhibits defects in its DNA helicase and DNA-dependent ATPase activities and was unable to support origin-specific DNA replicationin vivo. A kinetic analysis indicates that these defects are due to the inability of single-stranded DNA to induce high affinity ATP binding in UL9C111A protein. The DNA-dependent ATPase activity of UL9C111A protein is resistant toN-ethylmaleimide, while its DNA helicase activity remains sensitive. Accordingly, sensitivity of UL9 protein toN-ethylmaleimide is due to at least two cysteines. Cysteine 111 is involved in coupling single-stranded DNA binding to ATP-binding and subsequent hydrolysis, while a second cysteine is involved in coupling ATP hydrolysis to DNA unwinding.