Matsumoto K, Ishimi Y
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Mol Cell Biol. 1994 Jul;14(7):4624-32. doi: 10.1128/mcb.14.7.4624-4632.1994.
DNA unwinding of autonomously replicating sequence 1 (ARS1) from the yeast Saccharomyces cerevisiae was investigated. When a negatively supercoiled plasmid DNA containing ARS1 was digested with single-strand-specific mung bean nuclease, a discrete region in the vector DNA was preferentially digested. The regions containing the core consensus A domain and the 3'-flanking B domain of ARS1 were weakly digested. When the DNA was incubated with the multisubunit single-stranded DNA-binding protein (SSB, also called RPA [replication protein A]) from human and yeast cells prior to mung bean nuclease digestion, the cleavage in the A and B domains was greatly increased. Furthermore, a region corresponding to the 5'-flanking C domain of ARS1 was digested. These results indicate that three domains of ARS1, each of which is important for replication in yeast cells, closely correspond to the regions where the DNA duplex is easily unwound by torsional stress. SSB may stimulate the unwinding of the ARS1 region by its preferential binding to the destabilized three domains. Mung bean nuclease digestion of the substitution mutants with mutations of ARS1 (Y. Marahrens and B. Stillman, Science 255:817-823, 1992) revealed that the sequences in the B2 and A elements are responsible for the unwinding of the B domain and the region containing the A domain, respectively.
对酿酒酵母自主复制序列1（ARS1）的DNA解旋进行了研究。当用单链特异性绿豆核酸酶消化含有ARS1的负超螺旋质粒DNA时，载体DNA中的一个离散区域被优先消化。含有ARS1核心共有A结构域和3'侧翼B结构域的区域被微弱消化。当在绿豆核酸酶消化之前将DNA与人及酵母细胞的多亚基单链DNA结合蛋白（SSB，也称为RPA[复制蛋白A]）一起孵育时，A和B结构域的切割显著增加。此外，与ARS1的5'侧翼C结构域相对应的一个区域被消化。这些结果表明，ARS1的三个结构域，每个结构域对酵母细胞中的复制都很重要，紧密对应于DNA双链容易因扭转应力而解旋的区域。SSB可能通过其优先结合不稳定的三个结构域来刺激ARS1区域的解旋。对具有ARS1突变的替代突变体进行绿豆核酸酶消化（Y. Marahrens和B. Stillman，《科学》255:817 - 823，1992）表明，B2和A元件中的序列分别负责B结构域和包含A结构域区域的解旋。
