Studies on the mode of Ku interaction with DNA
Studies on the mode of Ku interaction with DNA
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DOI：
10.1074/jbc.m111916200
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发表时间：
2002-03-22
影响因子：
4.8
通讯作者：
Vindigni, A
中科院分区：
文献类型：
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作者：
Arosio, D;Cui, S;Vindigni, A
Ku异源二聚体在非同源末端连接中起核心作用。通过动态光散射、双滤光片结合、荧光光谱和带移测定研究了重组Ku与DNA的结合。Ku在溶液中的流体动力学半径为5.2 nm，并且当加入25-bp双链DNA（dsDNA）片段（1325）时不改变，表明只有一个Ku分子结合到25-bp片段。与D25结合的解离常数（k（d））为3.8 +/- 0.9 nm。如果底物的两端都用发夹环封闭，Ku仍然能够结合，k（d）变化很小。k（d）不受ATP、Mg 2+或离子强度的影响。然而，牛血清白蛋白的加入使kd降低了2倍。50 bp的DNA底物可以结合两个Ku分子，而三个分子结合到75 bp的底物。用Hill方程进行数据分析，得出Hill系数（n）值接近1，Ku与这些底物两端结合的kd值相同。因此，我们证明，有没有Ku异源二聚体结合较长的基板之间的合作相互作用。
The Ku heterodimer plays a central role in non-homologous end-joining. The binding of recombinant Ku to DNA has been investigated by dynamic light scattering, double-filter binding, fluorescence spectroscopy, and band shift assays. The hydrodynamic radius of Ku in solution is 5.2 nm and does not change when a 25-bp double-strand DNA (dsDNA) fragment (1325) is added, indicating that only one Ku molecule binds to a 25-bp fragment. The dissociation constant (k(d)) for the binding to D25 is 3.8 +/- 0.9 nm. If both ends of the substrate are closed with hairpin loops, Ku is still able to bind with little change in the k(d). The k(d) is not affected by ATP, Mg2+, or ionic strength. However, the addition of bovine serum albumin decreases the kd by 2-fold. DNA substrates of 50 bp can bind two Ku molecules, whereas three molecules are bound to a 75-bp substrate. Data analysis with the Hill equation yields a value of the Hill coefficient (n) close to 1, and the kd values for the binding of Ku to both ends of these substrates are the same. Thus, we demonstrate that there is no cooperative interaction among the Ku heterodimers binding longer substrates.