Engelman A, Hickman A B, Craigie R
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
J Virol. 1994 Sep;68(9):5911-7. doi: 10.1128/JVI.68.9.5911-5917.1994.
The integrase protein of human immunodeficiency virus type 1 removes two nucleotides from the 3' ends of reverse-transcribed human immunodeficiency virus type 1 DNA (3' processing) and covalently inserts the processed ends into a target DNA (DNA strand transfer). Mutant integrase proteins that lack the amino-and/or carboxyl-terminal domains are incapable of catalyzing 3' processing and DNA strand transfer but are competent for an apparent reversal of the DNA strand transfer reaction (disintegration) in vitro. Here, we investigate the binding of integrase to DNA by UV cross-linking. Cross-linked complexes form with a variety of DNA substrates independent of the presence of divalent metal ion. Analysis with amino- and carboxyl-terminal deletion mutant proteins shows that residues 213 to 266 of the 288-residue protein are required for efficient cross-linking in the absence of divalent metal ion. Carboxyl-terminal deletion mutants that lack this region efficiently cross-link only to the branched disintegration DNA substrate, and this reaction is dependent on the presence of metal ion. Both the core and C-terminal domains of integrase therefore contribute to nonspecific DNA binding.
1型人类免疫缺陷病毒的整合酶蛋白从逆转录的1型人类免疫缺陷病毒DNA的3'末端去除两个核苷酸（3'加工），并将加工后的末端共价插入目标DNA（DNA链转移）。缺乏氨基和/或羧基末端结构域的突变整合酶蛋白无法催化3'加工和DNA链转移，但在体外能够明显逆转DNA链转移反应（解离）。在此，我们通过紫外线交联研究整合酶与DNA的结合。交联复合物可与多种DNA底物形成，与二价金属离子的存在无关。对氨基和羧基末端缺失突变蛋白的分析表明，在不存在二价金属离子的情况下，288个氨基酸残基的蛋白中213至266位残基是有效交联所必需的。缺乏该区域的羧基末端缺失突变体仅能与分支解离DNA底物有效交联，且该反应依赖于金属离子的存在。因此，整合酶的核心结构域和C末端结构域均有助于非特异性DNA结合。
