Kennedy A K, Haniford D B, Mizuuchi K
Department of Biochemistry, University of Western Ontario, London, Canada.
Cell. 2000 Apr 28;101(3):295-305. doi: 10.1016/s0092-8674(00)80839-9.
The transposase family of proteins mediate DNA transposition or retroviral DNA integration via multistep phosphoryl transfer reactions. For Tn10 and phage Mu, a single active site of one transposase protomer catalyzes the successive transposition reaction steps. We examined phosphorothioate stereoselectivity at the scissile position for all four reaction steps catalyzed by the Tn10 transposase. The results suggest that the first three steps required for double-strand cutting at the transposon end proceed as a succession of pseudo-reverse reaction steps while the 3' end of the transposon remains bound to the same side of the active site. However, the mode of substrate binding to the active site changes for the cut transposon 3' end to target DNA strand joining. The phosphorothioate stereoselectivity of the corresponding steps of phage Mu transposition and HIV DNA integration matches that of Tn10 reaction, indicating a common mode of substrate-active site interactions for this class of DNA transposition reactions.
转座酶家族蛋白通过多步磷酸转移反应介导DNA转座或逆转录病毒DNA整合。对于Tn10和噬菌体Mu，一个转座酶原体的单个活性位点催化连续的转座反应步骤。我们研究了Tn10转座酶催化的所有四个反应步骤中切割位点的硫代磷酸酯立体选择性。结果表明，转座子末端双链切割所需的前三个步骤以一系列假逆反应步骤进行，而转座子的3'端仍与活性位点的同一侧结合。然而，对于切割后的转座子3'端与靶DNA链连接，底物与活性位点的结合模式发生了变化。噬菌体Mu转座和HIV DNA整合相应步骤的硫代磷酸酯立体选择性与Tn10反应的立体选择性相匹配，表明这类DNA转座反应存在底物-活性位点相互作用的共同模式。
