Peleg M, Kopel V, Borowiec J A, Manor H
Department of Biology, Technion-Israel Institute of Technology, Haifa.
Nucleic Acids Res. 1995 Apr 25;23(8):1292-9. doi: 10.1093/nar/23.8.1292.
Previous studies have indicated that d(TC)n.d(GA)n microsatellites may serve as arrest signals for mammalian DNA replication through the ability of such sequences to form DNA triple helices and thereby inhibit replication enzymes. To further test this hypothesis, we examined the ability of d(TC)i.d(GA)i.d(TC)i triplexes to inhibit DNA unwinding in vitro by a model eukaryotic DNA helicase, the SV40 large T-antigen. DNA substrates that were able to form triplexes, and non-triplex-forming control substrates, were tested. We found that the presence of DNA triplexes, as assayed by endonuclease S1 and osmium tetroxide footprinting, significantly inhibited DNA unwinding by T-antigen. Strong inhibition was observed not only at acidic pH values, in which the triplexes were most stable, but also at physiological pH values in the range 6.9-7.2. Little or no inhibition was detected at pH 8.7. Based on these results, and on previous studies of DNA polymerases, we suggest that DNA triplexes may form in vivo and cause replication arrest through a dual inhibition of duplex unwinding by DNA helicases and of nascent strand synthesis by DNA polymerases. DNA triplexes also have the potential to inhibit recombination and repair processes in which helicases and polymerases are involved.
先前的研究表明，d(TC)n.d(GA)n微卫星可能作为哺乳动物DNA复制的停滞信号，因为此类序列能够形成DNA三链螺旋，从而抑制复制酶。为了进一步验证这一假设，我们通过一种真核生物DNA解旋酶模型——SV40大T抗原，检测了d(TC)i.d(GA)i.d(TC)i三链体在体外抑制DNA解旋的能力。我们测试了能够形成三链体的DNA底物以及不能形成三链体的对照底物。我们发现，通过核酸酶S1和四氧化锇足迹法检测到的DNA三链体的存在，显著抑制了T抗原介导的DNA解旋。不仅在酸性pH值（此时三链体最稳定）下观察到了强烈的抑制作用，在6.9 - 7.2范围内的生理pH值下也观察到了强烈的抑制作用。在pH 8.7时几乎没有检测到抑制作用。基于这些结果以及先前对DNA聚合酶的研究，我们认为DNA三链体可能在体内形成，并通过双重抑制DNA解旋酶介导的双链解旋和DNA聚合酶介导的新生链合成来导致复制停滞。DNA三链体还有可能抑制涉及解旋酶和聚合酶的重组和修复过程。
