Interaction between the herpes simplex virus type 1 origin-binding and DNA polymerase accessory proteins.
Interaction between the herpes simplex virus type 1 origin-binding and DNA polymerase accessory proteins.
复制标题
1 型单纯疱疹病毒起源结合蛋白和 DNA 聚合酶辅助蛋白之间的相互作用。
DOI：
10.1006/viro.1997.8953
复制
发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Parris,DS
中科院分区：
文献类型：
--
作者：
Monahan,SJ;Grinstead,LA;Olivieri,W;Parris,DS
观察到单纯疱疹病毒1型(HSV-1)起源(ORI)结合蛋白(UL9)与功能DNA复制复合体的另外两个组成部分之间的相互作用。然而，到目前为止，UL9和DNA聚合酶全酶的一个组成部分之间还没有相互作用的证据。在这篇报道中，我们证明了UL9和DNA聚合酶辅助蛋白(UL42)可以在体外形成稳定的络合物，通过免疫共沉淀和谷胱甘肽-转移酶(GST)融合蛋白的亲和层析来确定。复合体的形成不需要其他病毒蛋白的存在，而是在溴化乙锭存在的情况下发生的，这表明UL9-UL42相互作用是DNA独立的。当GST-42融合蛋白浓度增加至5μM时，亲和小球可与兔网织红细胞裂解物体外转录/翻译中表达的UL9结合。UL9的N-末端和C-末端部分与GST亲和矩阵的结合表明，N-末端533个氨基酸足以与GST-42结合，尽管与全长蛋白质相比亲和力大约降低了4-6倍。没有观察到含有UL9C末端残基的多肽结合。因此，OrI结合蛋白UL9可以物理上与依赖于来源的DNA复制所需的每个复合体(解旋酶/启动酶、DNA聚合酶全酶、单链DNA结合蛋白)中的至少一个成员结合。这些特定的相互作用提供了一种方法，通过这种方法，HSV-1 DNA复制蛋白在复制起始处可以在感染细胞中有序组装，从而启动病毒DNA合成。
Interactions between the herpes simplex virus type 1 (HSV-1) origin (ori)-binding protein (UL9) and two other components of the functional DNA replication complex have been observed. However, to date, no interaction between UL9 and a component of the DNA polymerase holoenzyme has been demonstrated. In this report, we demonstrate that UL9 and the DNA polymerase accessory protein (UL42) can form a stable complexin vitroas determined by coimmunoprecipitation with specific antibodies to each protein and by affinity chromatography using glutathioneS-transferase (GST) fusion proteins. Complex formation does not require the presence of other viral proteins and occurs in the presence of ethidium bromide, indicating that UL9–UL42 interaction is DNA independent. Affinity beads charged with increasing concentrations of GST-42 fusion protein up to 5 μM bound increasing amounts of UL9 expressed byin vitrotranscription/translation in rabbit reticulocyte lysates. Binding of N- and C-terminal portions of UL9 to GST affinity matrices revealed that the N-terminal 533 amino acids were sufficient for binding to GST-42, albeit at approximately a four- to six-fold reduced affinity compared to the full-length protein. No binding of a polypeptide containing the remainder of the UL9 C-terminal residues was observed. Thus the ori-binding protein, UL9, can physically associate with at least one member of each of the complexes (helicase/primase, DNA polymerase holoenzyme, single-stranded DNA-binding protein) required for origin-dependent DNA replication. These specific interactions provide a means by which the ordered assembly of HSV-1 DNA replication proteins at origins of replication can occur in the infected cell for initiation of viral DNA synthesis.