Seki M, Enomoto T, Yanagisawa J, Hanaoka F, Ui M
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Biochemistry. 1988 Mar 8;27(5):1766-71. doi: 10.1021/bi00405a057.
The DNA helicase activity of DNA-dependent ATPase B purified from mouse FM3A cells [Seki, M., Enomoto, T., Hanaoka, F., & Yamada, M. (1987) Biochemistry 26, 2924-2928] has been further characterized. The helicase activity was assayed with partially duplex DNA substrates in which oligonucleotides to be released by the enzyme were radiolabeled. Oligonucleotides with or without phosphate at the 5' termini or with a deoxy- or dideoxyribose at the 3'-terminal nucleotides were displaced by this enzyme with essentially the same efficiency and with the same ATP (and dATP) and Mg2+ requirements. Thus, there was no strict structure requirement for both ends of duplex regions of substrates to be unwound by the enzyme. Shorter strands were released more readily than longer strands up to the length of 140 bases. The attachment of the enzyme to a single-stranded DNA region was a prerequisite for the neighboring duplex to be unwound; the enzyme-catalyzed unwinding was inhibited competitively by the coaddition of single-stranded DNAs which act as cofactors of the ATPase activity. Their activities as the inhibitor of helicase were well correlated with those as the cofactor of ATPase. The helicase B was found to migrate along single-stranded DNA in the 5' to 3' direction by the use of single strands with short duplex regions at both 3' and 5' ends as substrate. A possible role of this enzyme in DNA replication in mammalian cells is discussed.
从小鼠FM3A细胞中纯化得到的依赖DNA的ATP酶B的DNA解旋酶活性已得到进一步表征。使用部分双链DNA底物测定解旋酶活性，其中待酶释放的寡核苷酸被放射性标记。该酶以基本相同的效率以及相同的ATP（和dATP）与Mg2+需求置换5'端有或无磷酸基团、3'端核苷酸为脱氧核糖或双脱氧核糖的寡核苷酸。因此，对于酶要解开的底物双链区域的两端，不存在严格的结构要求。在长度达到140个碱基之前，较短的链比较长的链更容易被释放。酶附着于单链DNA区域是解开相邻双链的前提条件；作为ATP酶活性辅因子的单链DNA的共添加会竞争性抑制酶催化的解旋。它们作为解旋酶抑制剂的活性与作为ATP酶辅因子的活性密切相关。通过使用在3'和5'端均具有短双链区域的单链作为底物，发现解旋酶B沿单链DNA从5'向3'方向迁移。讨论了该酶在哺乳动物细胞DNA复制中的可能作用。
