Biswas N, Weller S K
Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
J Biol Chem. 2001 May 18;276(20):17610-9. doi: 10.1074/jbc.M010107200. Epub 2001 Jan 25.
Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains several conserved motifs found in primases; however, the contributions of each subunit to the biochemical activities of the subcomplex are not clear. In this work, the DNA binding properties of wild type and mutant subcomplexes were examined using single-stranded, duplex, and forked substrates. A gel mobility shift assay indicated that the UL5-UL52 subcomplex binds more efficiently to the forked substrate than to either single strand or duplex DNA. Although nucleotides are not absolutely required for DNA binding, ADP stimulated the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and adenosine 5'-O-(thiotriphosphate) stimulated the binding to a forked substrate. We have previously shown that both subunits contact single-stranded DNA in a photocross-linking assay (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked substrate when 5-iododeoxyuridine was located within the duplex portion. Photocross-linking experiments with subcomplexes containing mutant versions of UL5 and wild type UL52 indicated that the integrity of the ATP binding region is important for DNA binding of both subunits. These results support our previous proposal that UL5 and UL52 exhibit a complex interdependence for DNA binding (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active role in helicase activity than had previously been thought.
1型单纯疱疹病毒编码一种由UL5、UL52和UL8基因产物组成的异源三聚体解旋酶-引发酶复合物。UL5蛋白含有在解旋酶超家族1（SF1）的所有成员中都存在的七个基序，而UL52蛋白含有在引发酶中发现的几个保守基序；然而，每个亚基对亚复合物生化活性的贡献尚不清楚。在这项工作中，使用单链、双链和叉状底物检测了野生型和突变亚复合物的DNA结合特性。凝胶迁移率变动分析表明，UL5-UL52亚复合物与叉状底物的结合比与单链或双链DNA的结合更有效。虽然核苷酸对于DNA结合不是绝对必需的，但ADP刺激UL5-UL52与单链DNA的结合，而ATP、ADP和腺苷5'-O-（硫代三磷酸）刺激与叉状底物的结合。我们之前已经表明，在光交联分析中两个亚基都与单链DNA接触（Biswas，N.，和Weller，S.K.（1999）J.Biol.Chem.274，8068-8076）。在这项研究中，用叉状底物进行的光交联分析表明，UL5和UL52亚基在不同位置与叉状底物接触，UL52在单链DNA尾部，UL5在单链和双链DNA交界处附近。当5-碘脱氧尿苷位于双链部分时，两个亚基都不能与叉状底物交联。用含有UL5突变体版本和野生型UL52的亚复合物进行的光交联实验表明，ATP结合区域的完整性对于两个亚基的DNA结合都很重要。这些结果支持了我们之前的提议，即UL5和UL52在DNA结合方面表现出复杂的相互依赖性（Biswas，N.，和Weller，S.K.（1999）J.Biol.Chem.274，8068-8076），并表明UL52亚基在解旋酶活性中可能比之前认为的发挥更积极的作用。
