title: Resolution of Holliday junction recombination intermediates by wild-type and mutant IntDOT proteins.
abstract: 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.
authors.affiliation: Department of Microbiology, University of Illinois, B-103 Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801, USA.
authors.forename: Seyeun
authors.initials: S
authors.lastname: Kim
authors.forename: Jeffrey F
authors.initials: JF
authors.lastname: Gardner
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001439:Bacteroides / Q000201:enzymology*; D020137:Base Pair Mismatch; D004252:DNA Mutational Analysis; D045566:DNA, Cruciform / Q000378:metabolism*; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D045566:DNA, Cruciform; D050505:Mutant Proteins; D019426:Integrases