Single molecule detection of direct, homologous, DNA/DNA pairing
Single molecule detection of direct, homologous, DNA/DNA pairing
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DOI：
10.1073/pnas.0911214106
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发表时间：
2009-11-24
影响因子：
11.1
通讯作者：
Prentiss, M.
中科院分区：
文献类型：
--
作者：
Danilowicz, C.;Lee, C. H.;Prentiss, M.
使用一个平行的单分子磁镊分析，我们证明了同源配对的两个双链（DS）DNA分子的蛋白质，二价金属离子，拥挤剂，或游离的DNA末端的情况下。在温度和单价盐的生理条件下，配对是准确和快速的，即使在DNA分子浓度低于体内发现的那些数量级，并且在大量过量的非特异性竞争DNA的存在下。拥挤剂进一步提高反应速率。在5 kb或更大的同源性区域之间容易检测到配对。检测到的对在高达10 pN的热力和剪切力下是稳定的。这些结果强烈表明，直接识别化学完整的B-DNA分子之间的同源性应该是可能的，在体内。观察到的信号的鲁棒性提高了配对甚至可能是“默认”选项的可能性，仅限于特定特征所需的情况。理论上已经预测了完整dsDNA的蛋白质独立同源配对，但需要进一步的研究来确定现有的理论是否适合这里描述的序列长度，温度和盐的依赖性。
Using a parallel single molecule magnetic tweezers assay we demonstrate homologous pairing of two double-stranded (ds) DNA molecules in the absence of proteins, divalent metal ions, crowding agents, or free DNA ends. Pairing is accurate and rapid under physiological conditions of temperature and monovalent salt, even at DNA molecule concentrations orders of magnitude below those found in vivo, and in the presence of a large excess of nonspecific competitor DNA. Crowding agents further increase the reaction rate. Pairing is readily detected between regions of homology of 5 kb or more. Detected pairs are stable against thermal forces and shear forces up to 10 pN. These results strongly suggest that direct recognition of homology between chemically intact B-DNA molecules should be possible in vivo. The robustness of the observed signal raises the possibility that pairing might even be the "default" option, limited to desired situations by specific features. Protein-independent homologous pairing of intact dsDNA has been predicted theoretically, but further studies are needed to determine whether existing theories fit sequence length, temperature, and salt dependencies described here.