Kolakowski Adam J, Gardner Jeffrey F
Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, IL, USA.
Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, IL, USA.
Plasmid. 2016 Sep-Nov;87-88:10-16. doi: 10.1016/j.plasmid.2016.07.003. Epub 2016 Jul 12.
DOT Integrase (IntDOT) is a member of the tyrosine recombinase family. It catalyzes the integration and excision reactions of an integrative and conjugative element (ICE) called CTnDOT. Like other tyrosine recombinases, the integration reaction proceeds by two sets of strand exchanges between the attDOT site on CTnDOT and an attB site in the host chromosome. The strand exchanges occur seven bases apart and define an overlap region. After the first strand exchanges a Holliday Junction (HJ) intermediate is formed. Previous work showed that a valine (V95) in a predicted alpha helix in the N-terminus of IntDOT is required for resolution of HJs to substrates and products. We have identified two additional hydrophobic residues in the helix (A92 and F99) that are involved in resolution of HJs. IntDOT proteins with substitutions at these residues form aberrant complexes in an electrophoretic mobility shift assay. We propose that these three residues participate in hydrophobic interactions that are involved in forming higher-order complexes and resolution of HJs.
DOT整合酶（IntDOT）是酪氨酸重组酶家族的一员。它催化一种名为CTnDOT的整合性接合元件（ICE）的整合和切除反应。与其他酪氨酸重组酶一样，整合反应通过CTnDOT上的attDOT位点与宿主染色体上的attB位点之间的两组链交换进行。链交换相隔七个碱基，并定义了一个重叠区域。第一次链交换后形成一个霍利迪连接体（HJ）中间体。先前的研究表明，IntDOT N端预测的α螺旋中的一个缬氨酸（V95）对于将HJs分解为底物和产物是必需的。我们在该螺旋中鉴定出另外两个参与HJs分解的疏水残基（A92和F99）。在这些残基处有替代的IntDOT蛋白在电泳迁移率变动分析中形成异常复合物。我们提出这三个残基参与了形成高阶复合物和HJs分解所涉及的疏水相互作用。
