Requirement for a conserved serine in both processing and joining activities of retroviral integrase.
Requirement for a conserved serine in both processing and joining activities of retroviral integrase.
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逆转录病毒整合酶的加工和连接活动需要保守的丝氨酸。
DOI：
10.1073/pnas.89.15.6741
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发表时间：
1992
影响因子：
11.1
通讯作者：
Skalka,AM
中科院分区：
文献类型：
--
作者：
Katz,RA;Mack,JP;Merkel,G;Kulkosky,J;Ge,Z;Leis,J;Skalka,AM
逆转录病毒编码一种蛋白质，整合酶（IN），它是将病毒DNA插入宿主细胞染色体所必需的。IN可单独进行体外整合反应。该反应包括在两条病毒DNA链的3'末端附近进行核酸内切切割（处理步骤），然后将这些新的病毒DNA末端连接到宿主DNA上（连接步骤）。基于它们的进化保守性，我们先前已经确定了IN的至少11个氨基酸残基可能是反应所必需的。在这里，我们报告说，即使是保守的替代这些残基之一，不变的丝氨酸，产生严重的减少在两个加工和加入活动的劳斯肉瘤病毒在体外。1型人类免疫缺陷病毒IN的类似丝氨酸的替换对加工活性具有类似的影响。这些结果表明，这个单一的保守丝氨酸是活性位点的一个组成部分，一个活性位点用于加工和连接。用某些氨基酸取代该丝氨酸导致DNA结合活性的丧失或降低，而在该位置的其他取代似乎影响催化的后续步骤。所有有缺陷的劳斯肉瘤病毒IN都能够与野生型蛋白竞争，这支持了IN在多聚体复合物中发挥作用的模型。
Retroviruses encode a protein, the integrase (IN), that is required for insertion of the viral DNA into the host cell chromosome. IN alone can carry out the integration reaction in vitro. The reaction involves endonucleolytic cleavage near the 3' ends of both viral DNA strands (the processing step), followed by joining of these new viral DNA ends to host DNA (the joining step). Based on their evolutionary conservation, we have previously identified at least 11 amino acid residues of IN that may be essential for the reaction. Here we report that even conservative replacements of one of these residues, an invariant serine, produce severe reductions in both the processing and joining activities of Rous sarcoma virus IN in vitro. Replacement of the analogous serine of the type 1 human immunodeficiency virus IN had similar effects on processing activity. These results suggest that this single conserved serine is a component of the active site and that one active site is used for both processing and joining. Replacement of this serine with certain amino acids resulted in a loss or reduction in DNA binding activities, while other replacements at this position appeared to affect later steps in catalysis. All of the defective Rous sarcoma virus INs were able to compete with the wild-type protein, which supports a model in which IN functions in a multimeric complex.