Osman Fekret, Gaskell Louise, Whitby Matthew C
Department of Biochemistry, University of Oxford, Oxford, UK.
PLoS One. 2009;4(4):e5347. doi: 10.1371/journal.pone.0005347. Epub 2009 Apr 28.
Holliday junction (HJ) resolution is a critical step during homologous recombination. In Escherichia coli this job is performed by a member of the RNase H/Integrase superfamily called RuvC, whereas in Schizosaccharomyces pombe it has been attributed to the XPF family member Mus81-Eme1. HJ resolution is achieved through the sequential cleavage of two strands of like polarity at or close to the junction crossover point. RuvC functions as a dimer, whereas Mus81-Eme1 is thought to function as a dimer of heterodimers. However, in both cases the multimer contains two catalytic sites, which act independently and sequentially during the resolution reaction. To ensure that both strands are cleaved before the nuclease dissociates from the junction, the rate of second strand cleavage is greatly enhanced compared to that of the first. The enhancement of second strand cleavage has been attributed to the increased flexibility of the nicked HJ, which would facilitate rapid engagement of the second active site and scissile bond. Here we have investigated whether other properties of the nicked HJ are important for enhancing second strand cleavage.
A comparison of the efficiency of cleavage of nicked HJs with and without a 5' phosphate at the nick site shows that a 5' phosphate is required for most of the enhancement of second strand cleavage by RuvC. In contrast Mus81-Eme1 cleaves nicked HJs with and without a 5' phosphate with equal efficiency, albeit there are differences in cleavage site selection.
Our data show that efficient HJ resolution by RuvC depends on the 5' phosphate revealed by incision of the first strand. This is a hitherto unappreciated factor in promoting accelerated second strand cleavage. However, a 5' phosphate is not a universal requirement since efficient cleavage by Mus81-Eme1 appears to depend solely on the increased junction flexibility that is developed by the first incision.
霍利迪连接体（HJ）的解离是同源重组过程中的关键步骤。在大肠杆菌中，这项工作由核糖核酸酶H/整合酶超家族的成员RuvC完成，而在粟酒裂殖酵母中则归因于XPF家族成员Mus81-Eme1。HJ的解离是通过在连接交叉点处或其附近依次切割两条极性相同的链来实现的。RuvC以二聚体形式发挥作用，而Mus81-Eme1被认为以异源二聚体的二聚体形式发挥作用。然而，在这两种情况下，多聚体都包含两个催化位点，它们在解离反应过程中独立且依次起作用。为确保在核酸酶从连接体解离之前两条链都被切割，与第一条链切割相比，第二条链切割的速率大大提高。第二条链切割的增强归因于带切口的HJ灵活性增加，这将有助于第二个活性位点和可切割键的快速结合。在这里，我们研究了带切口的HJ的其他特性是否对增强第二条链切割很重要。
对切口位点有或没有5'磷酸基团的带切口HJ的切割效率进行比较表明，5'磷酸基团是RuvC增强第二条链切割的大部分作用所必需的。相比之下，Mus81-Eme1切割有或没有5'磷酸基团的带切口HJ的效率相同，尽管在切割位点选择上存在差异。
我们的数据表明，RuvC对HJ的有效解离取决于第一条链切割所暴露出的5'磷酸基团。这是促进第二条链加速切割中一个迄今未被认识到的因素。然而，5'磷酸基团并非普遍要求，因为Mus81-Eme1的有效切割似乎仅取决于第一个切口所产生的连接体灵活性增加。
