Shibata T, DasGupta C, Cunningham R P, Radding C M
Proc Natl Acad Sci U S A. 1979 Apr;76(4):1638-42. doi: 10.1073/pnas.76.4.1638.
Purified Escherichia coli recA protein catalyzed ATP-dependent pairing of superhelical DNA and homologous single-stranded fragments. The product of the reaction: (i) was retained by nitrocellulose filters in 1.5 M NaCl/0.15 M Na citrate at pH 7, (ii) was dissociated at pH 12.3 but was not dissociated by heating at 55 degrees C for 4 min or by treatment with 0.2% sodium dodecyl sulfate and proteinase K, (iii) contained covalently closed circular double-stranded DNA (form I DNA), (iv) contained single-stranded fragments associated with replicative form (RF) DNA, and (v) contained a significant fraction of D-loops as judged by electron microscopy. Linear and nicked circular double-stranded DNA did not substitute well for superhelical DNA; intact circular single-stranded DNA did not substitute well for single-stranded fragments. Homologous combinations of single-stranded fragments and superhelical DNA from phages phiX174 and fd reacted, whereas heterologous combinations did not. The reaction required high concentrations of protein and MgCl2. The ATPase activity of purified recA protein was more than 98% dependent on the addition of single-stranded DNA. In 1 mM MgCl2, the ability of superhelical DNA to support the ATPase activity was two-thirds as good as that of single-stranded DNA.
纯化的大肠杆菌recA蛋白催化超螺旋DNA与同源单链片段的ATP依赖性配对。反应产物：(i) 在pH 7的1.5 M NaCl/0.15 M柠檬酸钠中可被硝酸纤维素滤膜保留，(ii) 在pH 12.3时解离，但在55℃加热4分钟或用0.2%十二烷基硫酸钠和蛋白酶K处理时不解离，(iii) 包含共价闭合环状双链DNA（I型DNA），(iv) 包含与复制型（RF）DNA相关的单链片段，(v) 通过电子显微镜判断含有相当一部分D环。线性和带切口的环状双链DNA不能很好地替代超螺旋DNA；完整的环状单链DNA不能很好地替代单链片段。来自噬菌体φX174和fd的单链片段与超螺旋DNA的同源组合发生反应，而异源组合则不发生反应。该反应需要高浓度的蛋白质和MgCl2。纯化的recA蛋白的ATP酶活性超过98%依赖于单链DNA的添加。在1 mM MgCl2中，超螺旋DNA支持ATP酶活性的能力是单链DNA的三分之二。
