Characterization of a conjugative transposon integrase, IntDOT.
Sequence analysis revealed that the integrase of the Bacteroides conjugative transposon CTnDOT (IntDOT) might be a member of the tyrosine recombinase family because IntDOT has five of six highly conserved residues found in the catalytic domains of tyrosine recombinases. Yet, IntDOT catalyses a reaction that appears to differ in some respects from well-studied tyrosine recombinases such as that of phage lambda. To assess the importance of the conserved residues, we changed residues in IntDOT that align with conserved residues in tyrosine recombinases. Some substitutions resulted in a complete loss or significant decrease of integration activity in vivo. The ability of the mutant proteins to cleave and ligate CTnDOT attachment site (attDOT) DNA in vitro in general paralleled the in vivo results, but the H345A mutant, which had a wild-type level of integration in vivo, exhibited a slightly lower level of cleavage and ligation in vitro. Our results confirm the hypothesis that IntDOT belongs to the tyrosine recombinase family, but the catalytic core of the protein seems to have somewhat different organization. Previous DNA sequence analyses showed that CTnDOT att sites contain 5 bp non-homologous coupling sequences which were assumed to define the putative staggered sites of cleavage. However, cleavage assays showed that one of the cleavage sites is 2 bp away from the junction of CTnDOT and coupling sequence DNA. The site is in a region of homology that is conserved in CTnDOT att sites.
Malanowska
Karolina
K
Department of Microbiology, University of Illinois, Urbana, IL 61801, USA. malanows@uiuc.edu
Salyers
Abigail A
AA
Gardner
Jeffrey F
JF
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001439:Bacteroides / Q000235:genetics*; D001483:Base Sequence; D018797:Cell Cycle Proteins; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine / Q000378:metabolism
D001426:Bacterial Proteins; C089931:CDC36 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004251:DNA Transposable Elements; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; D004247:DNA; D019426:Integrases