Developing Single-Molecule TPM Experiments for Direct Observation of Successful RecA-Mediated Strand Exchange Reaction
Developing Single-Molecule TPM Experiments for Direct Observation of Successful RecA-Mediated Strand Exchange Reaction
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DOI：
10.1371/journal.pone.0021359
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发表时间：
2011-07-12
期刊：
影响因子：
3.7
通讯作者：
Li, Hung-Wen
中科院分区：
文献类型：
--
作者：
Fan, Hsiu-Fang;Cox, Michael M.;Li, Hung-Wen
RecA重组酶在同源重组中起核心作用。一旦组装在单链（ss）DNA上，RecA核蛋白丝介导同源DNA序列的配对和链交换过程。我们设计了两个基于系留粒子运动（TPM）的实验来研究E. coli RecA介导的配对和链交换在单分子水平上的力量的情况下。TPM实验测量由RecA结合和解离引起的指示DNA系链长度变化的系链珠布朗运动。在入侵链或输出链上标记的珠子的实验表明，在ATP或其不可水解的类似物ATP γ S存在下，DNA配对和链交换成功发生。链交换率和效率在ATP和ATP γ S条件下是相似的。此外，布朗运动的时间进程表明，链交换过程中进行单向的5 '到3'的方式，使用的突触段具有广泛和连续的大小分布。
RecA recombinases play a central role in homologous recombination. Once assembled on single-stranded (ss) DNA, RecA nucleoprotein filaments mediate the pairing of homologous DNA sequences and strand exchange processes. We have designed two experiments based on tethered particle motion (TPM) to investigate the fates of the invading and the outgoing strands during E. coli RecA-mediated pairing and strand exchange at the single-molecule level in the absence of force. TPM experiments measure the tethered bead Brownian motion indicative of the DNA tether length change resulting from RecA binding and dissociation. Experiments with beads labeled on either the invading strand or the outgoing strand showed that DNA pairing and strand exchange occurs successfully in the presence of either ATP or its non-hydrolyzable analog, ATP gamma S. The strand exchange rates and efficiencies are similar under both ATP and ATP gamma S conditions. In addition, the Brownian motion time-courses suggest that the strand exchange process progresses uni-directionally in the 5'-to-3' fashion, using a synapse segment with a wide and continuous size distribution.