The N-terminus of IntDOT forms hydrophobic interactions during Holliday Junction resolution

• Published in: Plasmid Vol. 87-88; pp. 10 - 16
• Main Authors: Kolakowski, Adam J., Gardner, Jeffrey F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2016
• Subjects: Bacteroides - genetics; Bacteroides - metabolism; Catalysis; Conjugation, Genetic; Conjugative transposon; CTnDOT; DNA, Cruciform - chemistry; DNA, Cruciform - genetics; Hydrophobic and Hydrophilic Interactions; Integrase; Integrases - chemistry; Integrases - genetics; Integrases - metabolism; Integrative and conjugative element; Mutation; Protein Binding; Protein Interaction Domains and Motifs; Integrase; Conjugative transposon; Integrative and conjugative element; CTnDOT
• ISSN: 0147-619X; 1095-9890
• DOI: 10.1016/j.plasmid.2016.07.003

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. •A hydrophobic pocket in H2 helix of IntDOT is identified.•This region participates in Holliday Junction resolution.•DNA cleavage and protein-protein interactions are also affected by this region.