Synapsis and DNA cleavage in φC31 integrase-mediated site-specific recombination
Synapsis and DNA cleavage in φC31 integrase-mediated site-specific recombination
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DOI：
10.1093/nar/gkh538
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发表时间：
2004-04-01
影响因子：
14.9
通讯作者：
Smith, MCM
中科院分区：
文献类型：
--
作者：
Smith, MCA;Till, R;Smith, MCM
链霉菌噬菌体phiC31编码一种整合酶，属于丝氨酸重组酶家族的定点重组酶。研究得很好的丝氨酸重组酶，即分解酶/转化酶，将两个重组位点聚集在一个突触中，然后催化DNA底物上协调一致的四链交错断裂，同时与凹陷的5‘端形成瞬时的共价连接。一对半位点相对于另一对半位点旋转180度，形成重组构型，然后连接DNA骨架。在这里，我们讨论了phiC31整合酶作用于其底物attP和attB时形成的重组中间体的性质。我们已经通过分析凝胶中的复合体以及这些复合体与蛋白酶处理后，鉴定了含有共价连接到切割的DNA底物attB或attP上的整合酶的中间体。利用催化失活的整合酶突变体S12A，鉴定了含有整合酶、attP和attB的突触复合体。此外，我们已经证明了含有插入或缺失的attB突变体在链切割阶段阻止了重组。因此，在attB内有严格的间距要求，可能是为了使催化丝氨酸相对于活性部位中的剪刀状磷酸正确定位。最后，利用整合酶S12A，我们证实了attL和AttR或其他位点的组合不能形成稳定的突触，表明整合重组的方向性是在突触处决定的。
The Streptomyces phage phiC31 encodes an integrase belonging to the serine recombinase family of site-specific recombinases. The well studied serine recombinases, the resolvase/invertases, bring two recombination sites together in a synapse, and then catalyse a concerted four-strand staggered break in the DNA substrates whilst forming transient covalent attachments with the recessed 5' ends. Rotation of one pair of half sites by 180degrees relative to the other pair occurs, to form the recombinant configuration followed by ligation of the DNA backbone. Here we address the nature of the recombination intermediates formed by phiC31 integrase when acting on its substrates attP and attB. We have identified intermediates containing integrase covalently attached to cleaved DNA substrates, attB or attP, by analysis of complexes in gels and after treatment of these complexes with proteinases. Using a catalytically inactive integrase mutant, S12A, the synaptic complexes containing integrase, attP and attB were identified. Furthermore, we have shown that attB mutants containing insertions or deletions are blocked in recombination at the stage of strand cleavage. Thus, there is a strict spacing requirement within attB, possibly for correct positioning of the catalytic serine relative to the scissile phosphate in the active site. Finally, using integrase S12A we confirmed the inability of attL and attR or other combinations of sites to form a stable synapse, indicating that the directionality of integrative recombination is determined at synapsis.