Bolt E L, Sharples G J, Lloyd R G
Institute of Genetics, University of Nottingham, Nottingham, NG7 2UH, UK.
J Mol Biol. 2000 Nov 24;304(2):165-76. doi: 10.1006/jmbi.2000.4196.
Holliday junctions are key intermediates in both homologous recombination and DNA repair, and are also formed from replication forks stalled at lesions in the template strands. Their resolution is critical for chromosome segregation and cell viability, and is mediated by a class of small, homodimeric endonucleases that bind the structure and cleave the DNA. All the enzymes studied require divalent metal ions for strand cleavage and their active centres are characterised by conserved aspartate/glutamate residues that provide ligands for metal binding. Sequence alignments reveal that they also contain a number of conserved basic residues. We used site-directed mutagenesis to investigate such residues in the RusA resolvase. RusA is a 120 amino acid residue polypeptide that can be activated in Escherichia coli to promote recombination and repair in the absence of the Ruv proteins. The RuvA, RuvB and RuvC proteins form a complex on Holliday junction DNA that drives coupled branch migration (RuvAB) and resolution (RuvC) reactions. In contrast to RuvC, the RusA resolvase does not interact directly with a branch migration motor, which simplifies analysis of its resolution activity. Catalysis depends on three highly conserved acidic residues (Asp70, Asp72 and Asp91) that define the catalytic centre. We show that Lys76, which is invariant in RusA sequences, is essential for catalysis, but not for DNA binding, and that an invariant asparagine residue (Asn73) is required for optimal activity. Analysis of DNA binding revealed that RusA may interact with one face of an open junction before manipulating its conformation in the presence of Mg(2+) as part of the catalytic process. A well-conserved arginine residue (Arg69) is linked with this critical stage. These findings provide the first insights into the roles played by basic residues in DNA binding and catalysis by a Holliday junction resolvase.
霍利迪连接体是同源重组和DNA修复过程中的关键中间体，也可由停滞在模板链损伤处的复制叉形成。它们的拆分对于染色体分离和细胞活力至关重要，并且由一类结合该结构并切割DNA的小型同二聚体内切核酸酶介导。所有研究过的酶进行链切割都需要二价金属离子，其活性中心的特征是具有保守的天冬氨酸/谷氨酸残基，这些残基为金属结合提供配体。序列比对显示，它们还含有一些保守的碱性残基。我们利用定点诱变来研究RusA拆分酶中的此类残基。RusA是一种由120个氨基酸残基组成的多肽，在大肠杆菌中可被激活，在没有Ruv蛋白的情况下促进重组和修复。RuvA、RuvB和RuvC蛋白在霍利迪连接体DNA上形成复合物，驱动耦合分支迁移（RuvAB）和拆分（RuvC）反应。与RuvC不同，RusA拆分酶不直接与分支迁移马达相互作用，这简化了对其拆分活性的分析。催化作用取决于定义催化中心的三个高度保守的酸性残基（天冬氨酸70、天冬氨酸72和天冬氨酸91）。我们发现，在RusA序列中不变的赖氨酸76对于催化作用至关重要，但对于DNA结合并非如此，并且一个不变的天冬酰胺残基（天冬酰胺73）是最佳活性所必需的。DNA结合分析表明，RusA可能在作为催化过程一部分在镁离子存在下操纵其构象之前，与开放连接体的一个面相互作用。一个保守性很好的精氨酸残基（精氨酸69）与这个关键阶段有关。这些发现首次揭示了碱性残基在霍利迪连接体拆分酶的DNA结合和催化过程中所起的作用。
