Motion of a DNA sliding clamp observed by single molecule fluorescence spectroscopy
Motion of a DNA sliding clamp observed by single molecule fluorescence spectroscopy
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DOI：
10.1074/jbc.m800174200
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发表时间：
2008-08-22
影响因子：
4.8
通讯作者：
Barsky, Daniel
中科院分区：
文献类型：
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作者：
Laurence, Ted A.;Kwon, Youngeun;Barsky, Daniel
DNA滑动夹附着在聚合酶上，并沿着DNA滑动，以允许DNA的快速、进行性复制。这些夹子含有许多带正电荷的残基，由于与带负电荷的DNA的吸引力相互作用，这些残基可以减少滑动。通过单分子光谱学，我们观察到一个荧光标记的滑动夹（聚合酶III β亚基或β夹）加载到自由扩散，单链M13环状DNA退火与荧光标记的DNA寡聚体高达90个碱基。我们发现β钳沿着DNA扩散的扩散常数约为10（-14）m（2）/s，比仅通过水扩散的扩散常数至少小3个数量级。我们还发现，在大肠杆菌单链结合蛋白的存在下，β钳仍然在3'端的证据。这些结果可能意味着钳不仅用于将聚合酶保持在DNA上，而且还防止沿着DNA的过度漂移沿着。
DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III beta subunit or beta clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the beta clamp diffusing along DNA is on the order of 10(-14) m(2)/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the beta clamp remains at the 3' end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA.