Lim H M, Lee H J, Jaxel C, Nadal M
Department of Biology, College of Natural Sciences, Chungnam National University, 305-764 Taejon, Republic of Korea.
J Biol Chem. 1997 Jul 18;272(29):18434-9. doi: 10.1074/jbc.272.29.18434.
Hin recombinase requires negatively supercoiled DNA for an efficient inversion. We have generated positively supercoiled plasmid DNA using reverse gyrase from Sulfolobus shibatae and subjected it to the Hin-mediated inversion reaction. Both Hin and Fis showed the same DNA binding activity regardless of the superhelical handedness of the substrate plasmid. However, inversion activity on positively supercoiled DNA was less than 1% of negatively supercoiled DNA. Assays designed to probe steps in inversion, showed that on positively supercoiled DNA, Hin was able to cleave the recombination sites with the same efficiency shown on negatively supercoiled DNA but was not able to exchange the cleaved DNA. Based on the theoretical differences between positive and negative supercoiling, our data may suggest that unwinding of the double helix at recombination sites is needed after DNA cleavage for strand exchange to occur.
Hin 重组酶需要负超螺旋 DNA 才能高效进行倒位。我们使用来自嗜热栖热菌的反向回旋酶生成了正超螺旋质粒 DNA，并使其进行 Hin 介导的倒位反应。无论底物质粒的超螺旋方向如何，Hin 和 Fis 都表现出相同的 DNA 结合活性。然而，正超螺旋 DNA 上的倒位活性不到负超螺旋 DNA 的 1%。旨在探测倒位步骤的实验表明，在正超螺旋 DNA 上，Hin 能够以与负超螺旋 DNA 相同的效率切割重组位点，但无法交换切割后的 DNA。基于正超螺旋和负超螺旋之间的理论差异，我们的数据可能表明，DNA 切割后，重组位点的双螺旋解旋对于链交换的发生是必要的。
