Malkov V A, Sastry L, Camerini-Otero R D
Genetics and Biochemistry Branch, National Institutes of Health (NIDDK), Building 10 Room 9D15, 10 Center Drive MSC 1810, Bethesda, MD, 20892-1810, USA.
J Mol Biol. 1997 Aug 15;271(2):168-77. doi: 10.1006/jmbi.1997.1164.
We have developed an in vitro selection procedure to elucidate the specificity of RecA assisted oligonucleotide recognition of double stranded DNA. The procedure was based on formation of a synaptic complex between an oligonucleotide-RecA filament and a supercoiled plasmid bearing a homologous partially degenerate region. The specificity of the selection depended on the reaction conditions: starting with a population that had, on average, 2.8 randomly distributed mismatches out of 27 bp, a population selected in the presence of 100 mM KCl had on average 1.0 mismatches, while a population selected at low ionic strength was less specific and had, on average, 2.0 mismatches. From the distributions of mismatches observed we calculated that the average destabilization free energy for one mismatch is 1.7(+/-0.5) kcal/mol. This is substantially less than the free energy for the incorporation of one mismatch in naked DNA duplex or a Py-Pu-Py triplex. Thus, RecA has an ability to decrease the fidelity of the homologous pairing reaction and minimize the cost of pairing between similar but not identical sequences. This "antiproofreading" activity of RecA protein does not require ATP hydrolysis.
我们开发了一种体外筛选程序，以阐明RecA辅助的寡核苷酸对双链DNA识别的特异性。该程序基于寡核苷酸-RecA细丝与带有同源部分简并区域的超螺旋质粒之间形成突触复合物。筛选的特异性取决于反应条件：从平均每27个碱基对中有2.8个随机分布错配的群体开始，在100 mM KCl存在下筛选的群体平均有1.0个错配，而在低离子强度下筛选的群体特异性较低，平均有2.0个错配。根据观察到的错配分布，我们计算出一个错配的平均去稳定自由能为1.7（±0.5）kcal/mol。这大大低于在裸DNA双链体或Py-Pu-Py三链体中掺入一个错配的自由能。因此，RecA具有降低同源配对反应保真度并最小化相似但不相同序列之间配对成本的能力。RecA蛋白的这种“反校对”活性不需要ATP水解。
