Divalent cations stimulate preferential recognition of a viral DNA end by HIV-1 integrase.
Divalent cations stimulate preferential recognition of a viral DNA end by HIV-1 integrase.
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DOI：
10.1021/bi982870n
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发表时间：
1999-06
期刊：
影响因子：
2.9
通讯作者：
J. Yi;E. Asante-Appiah;A. Skalka
中科院分区：
文献类型：
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作者：
J. Yi;E. Asante-Appiah;A. Skalka
在二价金属辅因子（Mg 2+或Mn 2+）的存在下，逆转录病毒编码的整合酶（IN）催化两种不同的反应：从病毒DNA的两个3'末端的两个核苷酸的位点特异性切割，以及凹陷的病毒末端与靶DNA中的交错磷酸的序列无关的连接。在这里，我们研究人类免疫缺陷病毒1型（HIV-1）的IN-DNA相互作用，使用表面等离子体共振。结果表明，IN与代表病毒DNA末端的双链体寡核苷酸和具有代表靶DNA底物的不相关序列的双链体寡核苷酸形成紧密复合物。IN-DNA复合物在4.0 M NaCl或50%（v/v）甲醇中是稳定的，但它们对低浓度的SDS没有抵抗力，这表明它们的稳定性高度依赖于蛋白质的结构特征。二价金属辅因子对IN-DNA相互作用产生两种不同的影响。Mn 2+抑制IN结合到模型靶DNA，在该阳离子存在下，表观Kd增加约3倍。另一方面，Mn 2+（或Mg 2+）刺激IN与模型病毒DNA末端的结合，使该IN-病毒DNA复合物的表观Kd降低约6倍。通过用GT/CA bp取代亚末端保守的CA/GT bp，IN与病毒DNA结合的这种金属介导的刺激被完全消除，并且当病毒DNA末端被凹陷或“预处理”时大大减少。IN结合到病毒双链体寡核苷酸，其末端用非病毒序列延伸，动力学类似于非病毒模型靶DNA。这表明在二价金属离子存在下，IN可以区分整合的DNA产物与病毒供体DNA。因此，我们的研究结果表明，HIV-1 IN对病毒DNA的优先识别仅在金属辅因子存在下实现，并且需要游离的野生型病毒DNA末端。
In the presence of a divalent metal cofactor (Mg2+ or Mn2+), retroviral-encoded integrase (IN) catalyzes two distinct reactions: site-specific cleavage of two nucleotides from both 3' ends of viral DNA, and sequence-independent joining of the recessed viral ends to staggered phosphates in a target DNA. Here we investigate human immunodeficiency virus type 1 (HIV-1) IN-DNA interactions using surface plasmon resonance. The results show that IN forms tight complexes both with duplex oligonucleotides that represent the viral DNA ends and with duplex oligonucleotides with an unrelated sequence that represent a target DNA substrate. The IN-DNA complexes are stable in 4.0 M NaCl, or 50% (v/v) methanol, but they are not resistant to low concentrations of SDS, indicating that their stability is highly dependent on structural features of the protein. Divalent metal cofactors exert two distinct effects on the IN-DNA interaction. Mn2+ inhibits IN binding to a model target DNA with the apparent Kd increasing approximately 3-fold in the presence of this cation. On the other hand, Mn2+ (or Mg2+) stimulates the binding of IN to a model viral DNA end, decreasing the apparent Kd of this IN-viral DNA complex approximately 6-fold. Such metal-mediated stimulation of the binding of IN to the viral DNA is totally abolished by substitution of the subterminal conserved CA/GT bp with a GT/CA bp, and is greatly diminished when the viral DNA end is recessed or "pre-processed." IN binds to a viral duplex oligonucleotide whose end was extended with nonviral sequences with kinetics similar to the nonviral model target DNA. This suggests that IN can distinguish the integrated DNA product from the viral donor DNA in the presence of divalent metal ion. Thus, our results show that preferential recognition of viral DNA by HIV-1 IN is achieved only in the presence of metal cofactor, and requires a free, wild-type viral DNA end.