RecA tests homology at both pairing and strand exchange
RecA tests homology at both pairing and strand exchange
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DOI：
10.1073/pnas.94.22.11863
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发表时间：
1997-10-28
影响因子：
11.1
通讯作者：
Radding, CM
中科院分区：
文献类型：
--
作者：
Bazemore, LR;FoltaStogniew, E;Radding, CM
RecA 是一种来自大肠杆菌的 38 kDa 蛋白质，可在单链 DNA 上聚合，形成核蛋白丝，与同源双链 DNA 配对并在体外进行链交换。为了观察错配对RecA催化重组反应动力学的影响，我们使用了基于荧光能量转移的测定法，可以区分配对和链置换阶段，测试了在链交换的异源双链体产物中产生2-14％错配的寡核苷酸序列，以及完全同源和异源序列。中间体的形成和解离速率的增加。此外，链置换率随着错配数量的增加而降低，大致与错配数量成正比。当异源性增加到 14% 时，配对的平衡常数和链置换的速率常数均下降 6 倍。这些结果表明，在配对和链交换过程中都发生同源性与异源性的区别。
RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. To observe the effects of mismatches on the kinetics of the RecA-catalyzed recombination reaction, we used assays based upon fluorescence energy transfer that can differentiate between the pairing and strand displacement phases, Oligonucleotide sequences that produced 2-14% mismatches in the heteroduplex product of strand exchange were tested, as well as completely homologous and heterologous sequences, The equilibrium constant for pairing decreased as the number of mismatches increased, which appeared to result from both a decrease in the rate of formation and an increase in the rate of dissociation of the intermediates. In addition, the rate of strand displacement decreased with increasing numbers of mismatches, roughly in proportion to the number of mismatches. The equilibrium constant for pairing and the rate constant for strand displacement both decreased 6-fold as the heterology increased to 14%, These results suggest that discrimination of homology from heterology occurs during both pairing and strand exchange.