INTERACTION OF HERPES-SIMPLEX VIRUS TYPE-1 DNA-POLYMERASE AND THE UL42 ACCESSORY PROTEIN WITH A MODEL PRIMER TEMPLATE
INTERACTION OF HERPES-SIMPLEX VIRUS TYPE-1 DNA-POLYMERASE AND THE UL42 ACCESSORY PROTEIN WITH A MODEL PRIMER TEMPLATE
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DOI：
10.1128/jvi.68.8.4937-4945.1994
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发表时间：
1994-08-01
影响因子：
5.4
通讯作者：
CHALLBERG, MD
中科院分区：
文献类型：
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作者：
GOTTLIEB, J;CHALLBERG, MD
遗传和生化研究表明，1 型单纯疱疹病毒 (HSV-1) DNA 聚合酶 (UL30) 和 UL42 基因的产物都是病毒 DNA 复制所必需的。先前的许多研究表明这两种蛋白质特异性相互作用，最近的研究证实来自 HSV-1 感染细胞的病毒 DNA 聚合酶由 UL30（Pol；催化亚基）和 UL42 多肽的异二聚体组成。 Pol-UL42 复合物与从重组杆状病毒感染的昆虫细胞中纯化的酶的分离亚基的催化特性的比较表明，Pol-UL42 复合物在单引物 M13 单链底物上比单独的 Pol 具有更高的持续性。这些研究的结果与 UL42 多肽是 HSV-1 DNA 聚合酶的辅助亚基的观点一致，其作用是提高聚合的持续性。初步实验表明，持续合成能力的增加伴随着聚合酶对线性双链 DNA 末端的亲和力的增加。我们进一步表征了 UL42 多肽对确定的发夹引物模板底物的影响。凝胶位移和填料结合研究表明，在 UL42 存在的情况下，Pol 催化亚基对引物模板 3' 末端的亲和力增加了 10 倍。 DNase I 足迹实验表明，Pol 催化亚基在保护 3' 双链体区域的 14 bp 和单链模板的相邻 18 个碱基的位置处与引物模板结合。 UL42多肽的存在导致双链体区域中连续5至14bp的额外保护，但不影响Pol亚基的5'位置。游离的 UL42 保护底物的整个双链区域，但不与单链区域结合。总而言之，这些结果表明，在 UL42 存在的情况下，持续合成能力的增加与游离 UL42 的双链 DNA 结合活性有关，并且 UL42 在 DNA 聚合酶复合物中的作用是充当钳子，降低聚合酶磨机在每个催化循环后从模板解离的可能性。
Genetic and biochemical studies have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication. A number of studies have previously suggested that these two proteins specifically interact, and more recent studies have confirmed that the viral DNA polymerase from HSV-1-infected cells consists of a heterodimer of the UL30 (Pol; the catalytic subunit) and UL42 polypeptides. A comparison of the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells indicated that the Pol-UL42 complex is more highly processive than Pol alone on singly primed M13 single-stranded substrates. The results of these studies are consistent with the idea that the UL42 polypeptide is an accessory subunit of the HSV-1 DNA polymerase that acts to increase the processivity of polymerization. Preliminary experiments suggested that the increase in processivity was accompanied by an increase in the affinity of the polymerase for the ends of linear duplex DNA. We have further characterized the effect of the UL42 polypeptide on a defined hairpin primer template substrate. Gel shift and filler binding studies show that the affinity of the Pol catalytic subunit for the 3' terminus of the primer template increases 10-fold in the presence of UL42. DNase I footprinting experiments indicate that the Pol catalytic subunit binds to the primer template at a position that protects 14 bp of the 3' duplex region and an adjacent 18 bases of the single-stranded template. The presence of the UL42 polypeptide results in the additional protection of a contiguous 5 to 14 bp in the duplex region but does not affect the 5' position of the Pol subunit. Free UL42 protects the entire duplex region of the substrate but does not bind to the single-stranded region. Taken together, these results suggest that the increase in processivity in the presence of UL42 is related to the double-stranded DNA-binding activity of free UL42 and that the role of UL42 in the DNA polymerase complex is to act as a clamp, decreasing the probability that the polymerase mill dissociate from the template after each cycle of catalysis.