Department of Physics, Harvard University, Cambridge, MA, USA.
Nucleic Acids Res. 2011 May;39(9):3781-91. doi: 10.1093/nar/gkr013. Epub 2011 Jan 18.
RecA is a key protein in homologous recombination. During recombination, one single-stranded DNA (ssDNA) bound to site I in RecA exchanges Watson-Crick pairing with a sequence-matched ssDNA that was part of a double-stranded DNA molecule (dsDNA) bound to site II in RecA. After strand exchange, heteroduplex dsDNA is bound to site I. In vivo, direct polymerization of RecA on dsDNA through site I does not occur, though it does in vitro. The mechanisms underlying the difference have been unclear. We use single-molecule experiments to decouple the two steps involved in polymerization: nucleation and elongation. We find that elongation is governed by a fundamental clock that is insensitive to force and RecA concentration from 0.2 and 6 µM, though rates depend on ionic conditions. Thus, we can probe nucleation site stability by creating nucleation sites at high force and then measuring elongation as a function of applied force. We find that in the presence of ATP hydrolysis a minimum force is required for polymerization. The minimum force decreases with increasing RecA or ATP concentrations. We propose that force reduces the off-rate for nucleation site binding and that nucleation site stability is the stringency factor that prevents in vivo polymerization.
RecA 是同源重组中的关键蛋白。在重组过程中，与 RecA 上的位点 I 结合的单链 DNA（ssDNA）与另一条单链 DNA 发生 Watson-Crick 配对，这条单链 DNA 是与 RecA 上的位点 II 结合的双链 DNA（dsDNA）的一部分。链交换后，异源双链 dsDNA 与位点 I 结合。在体内，dsDNA 通过位点 I 直接聚合的 RecA 不会发生，尽管在体外会发生。导致这种差异的机制尚不清楚。我们使用单分子实验将聚合反应涉及的两个步骤解耦：成核和延伸。我们发现，延伸由一个基本时钟控制，该时钟对来自 0.2 和 6 µM 的力和 RecA 浓度不敏感，尽管速率取决于离子条件。因此，我们可以通过在高力下创建成核位点，然后测量施加力下的延伸来探测成核位点的稳定性。我们发现，在 ATP 水解的情况下，聚合需要最小的力。最小力随 RecA 或 ATP 浓度的增加而减小。我们提出，力降低了成核位点结合的离解速率，而成核位点的稳定性是防止体内聚合的严格性因素。
