West S C, Parsons C A, Picksley S M
J Biol Chem. 1987 Sep 15;262(26):12752-8.
An endonuclease specific for cruciform junctions has been purified from yeast cells treated with a DNA-damaging agent. The activity was followed through five chromatographic steps by assaying for the linearization of supercoiled plasmid DNA, which extrudes cruciform structures in vitro. The sites of cleavage on pColIR215 were sequenced, and nicks were located to positions symmetrically opposed across the cruciform junction. The products of cleavage were unit length linear duplexes that contained terminal hairpin loops. In contrast to pColIR215, the cleavage patterns of pXG540 plasmid DNA were found to be complex, and cuts were found up to 40 bases from an (A-T)34 sequence that extrudes into a cruciform. Little or no activity could be detected on single-stranded DNA, linear duplex DNA, or nicked circular duplex DNA. The nuclease was insensitive to RNase but was inactivated by treatment with proteinase K. Mg2+ was required as cofactor and could not be replaced by Mn2+, Ca2+, Co2+, or Cu2+. The native molecular weight of the activity was approximately 200,000 as estimated by gel filtration.
一种对十字形结构具有特异性的核酸内切酶已从经DNA损伤剂处理的酵母细胞中纯化出来。通过检测超螺旋质粒DNA的线性化来跟踪该活性，超螺旋质粒DNA在体外会形成十字形结构，该活性经过五个色谱步骤。对pColIR215上的切割位点进行了测序，切口位于十字形结构相对的对称位置。切割产物是含有末端发夹环的单位长度线性双链体。与pColIR215不同，发现pXG540质粒DNA的切割模式很复杂，在一个延伸成十字形的(A-T)34序列上游40个碱基处都有切割。在单链DNA、线性双链DNA或带切口的环状双链DNA上几乎检测不到活性。该核酸酶对RNase不敏感，但用蛋白酶K处理会使其失活。需要Mg2+作为辅因子，且不能被Mn2+、Ca2+、Co2+或Cu2+替代。通过凝胶过滤估计，该活性的天然分子量约为200,000。
