Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China.
J Mol Cell Biol. 2010 Oct;2(5):264-75. doi: 10.1093/jmcb/mjq025.
Bacteriophage-encoded serine recombinases have great potential in genetic engineering but their catalytic mechanisms have not been adequately studied. Integration of ϕBT1 and ϕC31 via their attachment (att) sites is catalyzed by integrases of the large serine recombinase subtype. Both ϕBT1 and ϕC31 integrases were found to cleave single-substrate att sites without synaptic complex formation, and ϕBT1 integrase relaxed supercoiled DNA containing a single integration site. Systematic mutation of the central att site dinucleotide revealed that cleavage was independent of nucleotide sequence, but rejoining was crucially dependent upon complementarity of the cleavage products. Recombination between att sites containing dinucleotides with antiparallel complementarity led to antiparallel recombination. Integrase-substrate pre-incubation experiments revealed that the enzyme can form an attP-integrase tetramer complex that then captures naked attB DNA, and suggested that two alternative assembly pathways can lead to synaptic complex formation.
噬菌体编码的丝氨酸重组酶在基因工程中有很大的潜力，但它们的催化机制尚未得到充分研究。通过它们的附着（att）位点，φ BT1 和 φ C31 的整合是由大丝氨酸重组酶亚类的整合酶催化的。发现 φ BT1 和 φ C31 整合酶在没有突触复合物形成的情况下切割单底物 att 位点，并且 φ BT1 整合酶松弛含有单个整合位点的超螺旋 DNA。系统突变中心 att 位点二核苷酸显示，切割与核苷酸序列无关，但重新连接至关重要地取决于切割产物的互补性。含有具有反平行互补性的二核苷酸的 att 位点之间的重组导致反平行重组。整合酶-底物预孵育实验表明，该酶可以形成 attP-整合酶四聚体复合物，然后捕获裸露的 attB DNA，并表明两种替代的组装途径可以导致突触复合物的形成。
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