DeStefano J J, Bambara R A, Fay P J
Department of Biochemistry, University of Rochester, New York 14642.
Biochemistry. 1993 Jul 13;32(27):6908-15. doi: 10.1021/bi00078a014.
We have investigated the binding of human immunodeficiency virus reverse transcriptase (HIV-RT) to various hybrid RNA-DNA or DNA-DNA nucleic acid structures. Binding was measured by preequilibrating the RT with the nucleic acid substrate in the presence or absence of Mg2+ and then initiating synthesis or RNase H degradation reactions in the presence of excess "trap" polymer [poly(rA)-oligo(dT)]. The trap polymer sequestered RT molecules as soon as they dissociated from the substrate, such that the amount of synthesis or degradation on the substrate was proportional to the amount of bound RT. On hybrid substrates that had the 3' terminus of a complementary DNA oligomer recessed on a longer DNA or RNA template, binding to the RNA-DNA hybrid was more stable. Both the dissociation rate constant (k(off)) and equilibrium constant (Kd) values were larger for the DNA-DNA substrates by 5-10-fold. The difference was clearly in dissociation, since the association rate constant (k(on)) for both types of substrates was similar. On hybrid structures that had the 3' termini of a complementary RNA or DNA oligomer recessed on a longer DNA template, k(off) values are approximately the same on either structure. Although binding of the RT to DNA-DNA hybrid structures did not require Mg2+, its presence during the preequilibration period greatly stabilized binding. An approximate 20-60-fold decrease in the k(off), depending on the substrate structure, was observed with Mg2+. Measurements on one particular DNA-DNA hybrid indicated that the k(on) decreased by approximately 2 orders of magnitude with Mg2+. The relevance of these results to HIV replication is discussed.
我们研究了人类免疫缺陷病毒逆转录酶（HIV-RT）与各种RNA-DNA或DNA-DNA核酸杂交结构的结合情况。结合情况通过以下方式测定：在存在或不存在Mg2+的情况下，使RT与核酸底物预平衡，然后在过量“捕获”聚合物[聚（rA）-寡聚（dT）]存在下启动合成或RNase H降解反应。一旦RT分子从底物上解离，捕获聚合物就会将其隔离，使得底物上的合成或降解量与结合的RT量成正比。在互补DNA寡聚物的3'末端在较长的DNA或RNA模板上凹陷的杂交底物上，与RNA-DNA杂交体的结合更稳定。DNA-DNA底物的解离速率常数（k(off)）和平衡常数（Kd）值都大5-10倍。差异明显在于解离，因为两种类型底物的结合速率常数（k(on)）相似。在互补RNA或DNA寡聚物的3'末端在较长DNA模板上凹陷的杂交结构上，两种结构的k(off)值大致相同。虽然RT与DNA-DNA杂交结构的结合不需要Mg2+，但其在预平衡期的存在极大地稳定了结合。在Mg2+存在下，根据底物结构，观察到k(off)大约降低了20-60倍。对一种特定的DNA-DNA杂交体的测量表明，Mg2+存在时k(on)降低了大约2个数量级。讨论了这些结果与HIV复制的相关性。
