Chen Yei-Wei, Su Bo-Yu, Van Duyne Gregory D, Fogg Paul, Fan Hsiu-Fang
Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, Taiwan; Department of Chemistry, National Sun Yat-sen University, Kaohsiung, Taiwan; Aerosol Science Research Center, National Sun Yat-sen University, Kaohsiung, Taiwan.
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan.
Biophys J. 2023 Dec 19;122(24):4656-4669. doi: 10.1016/j.bpj.2023.11.009. Epub 2023 Nov 16.
Serine integrases promote the recombination of two complementary DNA sequences, attP and attB, to create hybrid sequences, attL and attR. The reaction is unidirectional in the absence of an accessory protein called recombination directionality factor. We utilized tethered particle motion (TPM) experiments to investigate the reaction behaviors of two model serine integrases from Listeria innocua phage LI and Streptomyces coelicolor phage C31. Detailed kinetic analyses of wild-type and mutant proteins were carried out to verify the mechanisms of recombination directionality. In particular, we assessed the influence of a coiled-coil motif (CC) that is conserved in the C-terminal domain of serine integrases and is an important prerequisite for efficient recombination. Compared to wild type, we found that CC deletions in both serine integrases reduced the overall abundance of integrase (Int) att-site complexes and favored the formation of nonproductive complexes over recombination-competent complexes. Furthermore, the rate at which CC mutants formed productive synaptic complexes and disassembled aberrant nonproductive complexes was significantly reduced. It is notable that while the φC31 Int CC is essential for recombination, the LI Int CC plays an auxiliary role for recombination to stabilize protein-protein interactions and to control the directionality of the reaction.
丝氨酸整合酶促进两个互补DNA序列attP和attB的重组，以产生杂合序列attL和attR。在没有一种称为重组方向性因子的辅助蛋白的情况下，该反应是单向的。我们利用系留粒子运动（TPM）实验来研究来自无害李斯特菌噬菌体LI和天蓝色链霉菌噬菌体C31的两种模型丝氨酸整合酶的反应行为。对野生型和突变型蛋白进行了详细的动力学分析，以验证重组方向性的机制。特别地，我们评估了丝氨酸整合酶C端结构域中保守的卷曲螺旋基序（CC）的影响，它是高效重组的重要前提条件。与野生型相比，我们发现两种丝氨酸整合酶中的CC缺失均降低了整合酶（Int）att位点复合物的总体丰度，并且相比于有重组能力的复合物，更有利于非生产性复合物的形成。此外，CC突变体形成有生产性的突触复合物和解离异常非生产性复合物的速率显著降低。值得注意的是，虽然φC31 Int CC对重组至关重要，但LI Int CC在重组中起辅助作用，以稳定蛋白质-蛋白质相互作用并控制反应的方向性。
