Freeman Alasdair D J, Liu Yijin, Déclais Anne-Cécile, Gartner Anton, Lilley David M J
Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dow Street, Dundee DD1 5EH, UK.
J Mol Biol. 2014 Dec 12;426(24):3946-3959. doi: 10.1016/j.jmb.2014.10.008. Epub 2014 Oct 12.
Processing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1nt 3' to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1.
霍利迪连接体的加工在重组过程中至关重要。我们已经从嗜热真菌嗜热毛壳菌中鉴定出连接体解析酶GEN1的基因，并表达了其N端487个氨基酸的片段。该蛋白是一种核酸酶，对四链DNA连接体具有高度选择性，在围绕对角轴对称排列的两条链上，在链交换点的3'端切割1个核苷酸。CtGEN1以具有纳摩尔亲和力的离散同型二聚体形式与DNA连接体结合。对十字形切割动力学的分析表明，第二条链的切割比第一次切割快一个数量级，从而产生有效的解析事件。所有这些特性与细菌、噬菌体和线粒体连接体解析酶所描述的特性非常相似。CtGEN1在特性上也与人类酶相似，但没有目前阻碍对后者蛋白进行详细分析的聚集问题。因此，CtGEN1是用于真核生物GEN1生物物理和结构分析的优秀酶。
