Assembly and catalytic properties of retrovirus integrase-DNA complexes capable of efficiently performing concerted integration.
Assembly and catalytic properties of retrovirus integrase-DNA complexes capable of efficiently performing concerted integration.
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逆转录病毒整合酶-DNA复合物的组装和催化特性能够有效地进行协同整合。
DOI：
10.1128/jvi.69.12.7483-7488.1995
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Grandgenett,DP
中科院分区：
文献类型：
--
作者：
Vora,AC;Grandgenett,DP
研究了含有线性逆转录病毒样DNA和整合酶（in）的核蛋白复合物的体外组装过程。建立了禽成髓细胞病病毒IN有效配对两个独立的线性DNA片段（每个长度为487 bp）的溶液条件，其中包含3' OH嵌入的长末端重复末端。在冰上预孵育期间，将病毒末端与IN配对，允许这些核蛋白复合物催化两个末端协同插入环状DNA靶标（全位点反应），模拟体内反应。三个主要的溶液决定因素是高浓度的NaCl （0.33 M）、1,4-二氧六环和聚乙二醇。非质子溶剂二氧六环（15%）明显优于15%二甲亚砜（6倍）形成的配合物，能够形成全位点整合事件而不是半位点整合事件。IN的半位点反应涉及将单个供体末端插入环状pGEM。虽然NaCl是有效促进协同整合反应的必要条件，但在高NaCl浓度下，二氧六环是防止半位反应发生的必要条件。在最佳溶液条件下，协同积分反应与IN的6倍范围成正比。复合物似乎不会翻转，并且很少产生半位点供体-供体分子。在0.15或0.35 M NaCl的存在下，二氧六环阻止了In对钝端供体的3' OH有效修剪，这表明In与钝端供体形成的配合物与含有3' OH嵌入末端的供体形成的配合物不同。结果表明，单用IN能够有效促进体外协同整合反应的蛋白-蛋白和蛋白- dna相互作用。
The in vitro assembly process for forming nucleoprotein complexes containing linear retrovirus-like DNA and integrase (IN) was investigated. Solution conditions that allowed avian myeloblastosis virus IN to efficiently pair two separate linear DNA fragments (each 487 bp in length) containing 3' OH recessed long terminal repeat termini were established. Pairing of the viral termini by IN during preincubation on ice permitted these nucleoprotein complexes to catalyze the concerted insertion of the two termini into a circular DNA target (full-site reaction), mimicking the in vivo reaction. The three major solution determinants were high concentrations of NaCl (0.33 M), 1,4-dioxane, and polyethylene glycol. The aprotic solvent dioxane (15%) was significantly better (sixfold) than 15% dimethyl sulfoxide for forming complexes capable of full-site rather than half-site integration events. Half-site reactions by IN involved the insertion of a single donor terminus into circular pGEM. Although NaCl was essential for the efficient promotion of the concerted integration reaction, dioxane was necessary to prevent half-site reactions from occurring at high NaCl concentrations. Under optimal solution conditions, the concerted integration reaction was directly proportional to a sixfold range of IN. The complexes appeared not to turn over, and few half-site donor-donor molecules were produced. In the presence of 0.15 or 0.35 M NaCl, dioxane prevented efficient 3' OH trimming of a blunt-ended donor by IN, suggesting that the complexes formed by IN with blunt-ended donors were different from those formed with donors containing 3' OH recessed termini for strand transfer. The results suggest that IN alone was capable of protein-protein and protein-DNA interactions that efficiently promote the in vitro concerted integration reaction.