Benson F E, West S C
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, United Kingdom.
J Biol Chem. 1994 Feb 18;269(7):5195-201.
The specificity of the Escherichia coli RuvC Holliday junction resolvase has been investigated in vitro. RuvC protein cleaves synthetic DNA substrates that model three- or four-stranded recombination intermediates but fails to act upon Y junctions, G/A mismatches, heterologous loop structures, or two-stranded branched junctions. RuvC therefore differs from endonuclease VII of bacteriophage T4 which exhibits broad range specificity. Using related three- and four-stranded synthetic DNA junctions, we show that RuvC cleaves both junctions at the same DNA sequence and requires a region of homology at the junction point. The action of RuvC on three- and four-stranded recombination intermediates made by RecA was also investigated. We found that RuvC fails to resolve three-stranded intermediates in the presence of RecA, although four-stranded intermediates are resolved under the same conditions. However, both three- and four-stranded intermediates are substrates for the nuclease after removal of RecA. We interpret these differences in terms of the contiguity of the RecA nucleoprotein filament which may, under certain conditions, limit access to the Holliday junction resolvase.
已在体外研究了大肠杆菌RuvC霍利迪连接体解离酶的特异性。RuvC蛋白可切割模拟三链或四链重组中间体的合成DNA底物，但对Y型连接、G/A错配、异源环结构或双链分支连接不起作用。因此，RuvC不同于噬菌体T4的内切核酸酶VII，后者具有广泛的特异性。使用相关的三链和四链合成DNA连接体，我们发现RuvC在相同的DNA序列处切割这两种连接体，并且在连接点处需要一段同源区域。还研究了RuvC对由RecA形成的三链和四链重组中间体的作用。我们发现，在RecA存在的情况下，RuvC无法解离三链中间体，尽管在相同条件下四链中间体能被解离。然而，去除RecA后，三链和四链中间体都是该核酸酶的底物。我们根据RecA核蛋白丝的连续性来解释这些差异，在某些条件下，RecA核蛋白丝可能会限制对霍利迪连接体解离酶的接近。
