Department of Biochemistry, Molecular biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Nucleic Acids Res. 2013 Jan 7;41(1):648-56. doi: 10.1093/nar/gks1015. Epub 2012 Oct 31.
Holliday junction (HJ) resolvases are structure-specific endonucleases that cleave four-way DNA junctions (HJs) generated during DNA recombination and repair. Bacterial RuvC, a prototypical HJ resolvase, functions as homodimer and nicks DNA strands precisely across the junction point. To gain insights into the mechanisms underlying symmetrical strand cleavages by RuvC, we performed crystallographic and biochemical analyses of RuvC from Thermus thermophilus (T.th. RuvC). The crystal structure of T.th. RuvC shows an overall protein fold similar to that of Escherichia coli RuvC, but T.th. RuvC has a more tightly associated dimer interface possibly reflecting its thermostability. The binding mode of a HJ-DNA substrate can be inferred from the shape/charge complementarity between the T.th. RuvC dimer and HJ-DNA, as well as positions of sulfate ions bound on the protein surface. Unexpectedly, the structure of T.th. RuvC homodimer refined at 1.28 Å resolution shows distinct asymmetry near the dimer interface, in the region harboring catalytically important aromatic residues. The observation suggests that the T.th. RuvC homodimer interconverts between two asymmetric conformations, with alternating subunits switched on for DNA strand cleavage. This model provides a structural basis for the 'nick-counter-nick' mechanism in HJ resolution, a mode of HJ processing shared by prokaryotic and eukaryotic HJ resolvases.
霍利迪连接点（HJ）解旋酶是一种结构特异性内切酶，可切割 DNA 重组和修复过程中产生的四链 DNA 连接点（HJ）。细菌 RuvC 是一种典型的 HJ 解旋酶，作为同源二聚体发挥作用，并在连接点精确地切割 DNA 链。为了深入了解 RuvC 对称链切割的机制，我们对来自嗜热栖热菌（T.th. RuvC）的 RuvC 进行了晶体学和生化分析。T.th. RuvC 的晶体结构显示出与大肠杆菌 RuvC 相似的整体蛋白折叠，但 T.th. RuvC 的二聚体界面更紧密，可能反映了其热稳定性。通过 T.th. RuvC 二聚体与 HJ-DNA 之间的形状/电荷互补性以及结合在蛋白质表面上的硫酸盐离子的位置，可以推断出 HJ-DNA 底物的结合模式。出乎意料的是，在分辨率为 1.28 Å 的条件下，T.th. RuvC 同源二聚体的结构显示出在二聚体界面附近存在明显的不对称性，该区域含有催化重要的芳香族残基。该观察结果表明，T.th. RuvC 同源二聚体在两个不对称构象之间相互转换，用于 DNA 链切割的亚基交替开启。该模型为 HJ 分辨率中的“切口-对切口”机制提供了结构基础，这是一种原核和真核 HJ 解旋酶共享的 HJ 加工模式。
