Jacoby D B, Wensink P C
Rosenstiel Center, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
J Biol Chem. 1996 Jul 12;271(28):16827-32. doi: 10.1074/jbc.271.28.16827.
YPF1, a heterodimeric protein from Drosophila melanogaster, is a homolog to Ku, the DNA binding subunit of human DNA-dependent protein kinase. This kinase is crucial in transcriptional activation, V(D)J recombination, double-strand break repair, and both topoisomerase and helicase activities. To investigate functional homology between YPF1 and Ku, we examined DNA binding properties of YPF1. Like Ku, at 100 mM KCl, YPF1 binding has no detectable DNA sequence specificity, requires a DNA terminus, and has a concentration-dependent stoichiometry consistent with subsequent translocation along DNA. YPF1 differs from Ku by having a 10(5)-fold higher affinity. At 400 mM KCl, YPF1 still prefers DNA termini but shows binding specificities not observed previously with Ku. In descending order of affinity, YPF1 binds to: specific DNA sequences with a specific polarity and spacing relative to DNA termini; nonspecific linear DNA; and circular DNA. At this higher ionic strength, binding stoichiometry is concentration independent, indicating that YPF1 remains bound to ends. These results demonstrate a strong functional homology between YPF1 and Ku at physiological ionic strength. The strong binding of YPF1 has also allowed us to detect underlying binding specificities that may be specific to YPF1 and its function.
YPF1是一种来自黑腹果蝇的异源二聚体蛋白，是人类DNA依赖性蛋白激酶的DNA结合亚基Ku的同源物。这种激酶在转录激活、V(D)J重组、双链断裂修复以及拓扑异构酶和解旋酶活性中都至关重要。为了研究YPF1和Ku之间的功能同源性，我们检测了YPF1的DNA结合特性。与Ku一样，在100 mM KCl条件下，YPF1的结合没有可检测到的DNA序列特异性，需要DNA末端，并且具有与随后沿DNA易位一致的浓度依赖性化学计量。YPF1与Ku的不同之处在于其亲和力高10^5倍。在400 mM KCl条件下，YPF1仍然偏爱DNA末端，但显示出以前在Ku中未观察到的结合特异性。按亲和力从高到低的顺序，YPF1与以下物质结合：相对于DNA末端具有特定极性和间距的特定DNA序列；非特异性线性DNA；以及环状DNA。在这种较高的离子强度下，结合化学计量与浓度无关，表明YPF1仍然与末端结合。这些结果证明了在生理离子强度下YPF1和Ku之间存在很强的功能同源性。YPF1的强结合也使我们能够检测到可能特定于YPF1及其功能的潜在结合特异性。
