Department of Microbiology, University of Illinois, B-103 Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801, USA.
J Bacteriol. 2011 Mar;193(6):1351-8. doi: 10.1128/JB.01465-10. Epub 2011 Jan 7.
CTnDOT encodes an integrase that is a member of the tyrosine recombinase family. The recombination reaction proceeds by sequential sets of genetic exchanges between the attDOT site in CTnDOT and an attB site in the chromosome. The exchanges are separated by 7 base pairs in each site. Unlike most tyrosine recombinases, IntDOT exchanges sites that contain different DNA sequences between the exchange sites to generate Holliday junctions (HJs) that contain mismatched bases. We demonstrate that IntDOT resolves synthetic HJs in vitro. Holliday junctions that contain identical sequences between the exchange sites are resolved into both substrates and products, while HJs that contain mismatches are resolved only to substrates. This result implies that resolution of HJs to products requires the formation of a higher-order nucleoprotein complex with natural sites containing IntDOT. We also found that proteins with substitutions of residues (V95, K94, and K96) in a putative alpha helix at the junction of the N and CB domains (coupler region) were defective in resolving HJs. Mutational analysis of charged residues in the coupler and the N terminus of the protein did not provide evidence for a charge interaction between the regions of the protein. V95 may participate in a hydrophobic interaction with another region of IntDOT.
CTnDOT 编码一种整合酶，属于酪氨酸重组酶家族。重组反应通过 CTnDOT 中的 attDOT 位点和染色体上的 attB 位点之间的一系列遗传交换来进行。每个位点的交换由 7 个碱基对分隔开。与大多数酪氨酸重组酶不同，IntDOT 在交换位点之间交换含有不同 DNA 序列的位点，从而产生含有错配碱基的 Holliday 连接点 (HJ)。我们证明了 IntDOT 可以在体外解析合成的 HJ。在交换位点之间含有相同序列的 HJ 被解析为底物和产物，而含有错配的 HJ 仅被解析为底物。这个结果意味着，HJ 到产物的解析需要形成具有自然位点的更高阶核蛋白复合物，这些自然位点含有 IntDOT。我们还发现，在 N 和 CB 结构域（连接区）交界处的一个假定α螺旋中的残基（V95、K94 和 K96）发生取代的蛋白在解析 HJ 方面存在缺陷。对 coupler 区域和蛋白 N 末端的带电残基进行突变分析并没有为蛋白的这些区域之间的电荷相互作用提供证据。V95 可能与 IntDOT 的另一个区域参与疏水相互作用。
