Castella Sandrine, Burgin David, Sanders Cyril M
Institute for Cancer studies, University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK.
Nucleic Acids Res. 2006 Aug 7;34(13):3731-41. doi: 10.1093/nar/gkl554. Print 2006.
The E1 protein of bovine papillomavirus type-1 is the viral replication initiator protein and replicative helicase. Here we show that the C-terminal approximately 300 amino acids of E1, that share homology with members of helicase superfamily 3 (SF3), can act as an autonomous helicase. E1 is monomeric in the absence of ATP but assembles into hexamers in the presence of ATP, single-stranded DNA (ssDNA) or both. A 16 base sequence is the minimum for efficient hexamerization, although the complex protects approximately 30 bases from nuclease digestion, supporting the notion that the DNA is bound within the protein complex. In the absence of ATP, or in the presence of ADP or the non-hydrolysable ATP analogue AMP-PNP, the interaction with short ssDNA oligonucleotides is exceptionally tight (T(1/2) > 6 h). However, in the presence of ATP, the interaction with DNA is destabilized (T(1/2) approximately 60 s). These results suggest that during the ATP hydrolysis cycle an internal DNA-binding site oscillates from a high to a low-affinity state, while protein-protein interactions switch from low to high affinity. This reciprocal change in protein-protein and protein-DNA affinities could be part of a mechanism for tethering the protein to its substrate while unidirectional movement along DNA proceeds.
牛乳头瘤病毒1型的E1蛋白是病毒复制起始蛋白和复制解旋酶。我们在此表明，E1蛋白C端约300个氨基酸与解旋酶超家族3（SF3）成员具有同源性，可作为自主解旋酶发挥作用。在无ATP时E1呈单体状态，但在有ATP、单链DNA（ssDNA）或两者同时存在时会组装成六聚体。16个碱基序列是高效形成六聚体的最小长度，尽管该复合物可保护约30个碱基不被核酸酶消化，这支持了DNA结合在蛋白质复合物内的观点。在无ATP时，或在有ADP或不可水解的ATP类似物AMP-PNP存在时，与短ssDNA寡核苷酸的相互作用异常紧密（半衰期>6小时）。然而，在有ATP时，与DNA的相互作用不稳定（半衰期约60秒）。这些结果表明，在ATP水解循环中，一个内部DNA结合位点从高亲和力状态振荡到低亲和力状态，而蛋白质-蛋白质相互作用则从低亲和力切换到高亲和力。蛋白质-蛋白质和蛋白质-DNA亲和力的这种相互变化可能是在沿DNA单向移动时将蛋白质拴系到其底物上的机制的一部分。
