Allingham John S, Haniford David B
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5B7.
J Mol Biol. 2002 May 24;319(1):53-65. doi: 10.1016/S0022-2836(02)00297-8.
Tn10/IS10 transposition involves assembly of a synaptic complex (or transpososome) in which two transposon ends are paired, followed by four distinct chemical steps at each transposon end. The chemical steps are dependent on the presence of a suitable divalent metal cation (Me(2+)). Transpososome assembly and structure are also affected by Me(2+). To gain further insight into the mechanisms of Me(2+) action in Tn10/IS10 transposition we have investigated the effects of substituting Mn(2+) for Mg(2+), the physiologic Me(2+), in transposition. We have also investigated the significance of an Me(2+)-assisted conformational change in transpososome structure. We show that Mn(2+) has two previously unrecognized effects on the Tn10 donor cleavage reaction. It accelerates the rates of hairpin formation and hairpin resolution without significantly affecting the rate of the first chemical step, first strand nicking. Mn(2+) also relaxes the specificity of first strand nicking. We also show that Me(2+)-assisted transpososome unfolding coincides with a structural transition in the transposon-donor junction that may be necessary for hairpin formation. Possible mechanisms for these observations are considered.
Tn10/IS10转座涉及一个突触复合体（或转座体）的组装，其中两个转座子末端配对，随后在每个转座子末端进行四个不同的化学步骤。这些化学步骤依赖于合适的二价金属阳离子（Me(2+)）的存在。转座体的组装和结构也受Me(2+)影响。为了进一步深入了解Me(2+)在Tn10/IS10转座中的作用机制，我们研究了用Mn(2+)替代生理状态下的Me(2+)即Mg(2+)对转座的影响。我们还研究了Me(2+)辅助的转座体构象变化在转座体结构中的重要性。我们发现Mn(2+)对Tn10供体切割反应有两个先前未被认识到的影响。它加速了发夹形成和发夹解离的速率，而对第一个化学步骤即第一链切口的速率没有显著影响。Mn(2+)还放宽了第一链切口的特异性。我们还表明，Me(2+)辅助的转座体解折叠与转座子-供体连接处的结构转变同时发生，这可能是发夹形成所必需的。文中考虑了这些观察结果的可能机制。
