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Concerted action of three distinct domains in the DNA cleaving-joining reaction catalyzed by relaxase (TraI) of conjugative plasmid RP4
- Source :
- The Journal of biological chemistry. 269(4)
- Publication Year :
- 1994
-
Abstract
- The TraI protein of plasmid RP4 (IncP alpha) catalyzes a site- and strand-specific cleaving-joining reaction on form I or single-stranded DNA. Thus, TraI is one of the key components involved in the initiation and termination of horizontal DNA transfer by bacterial conjugation. Amino acid sequence comparison revealed three motifs in the TraI sequence conserved in relaxases from different origins. Site-directed mutagenesis of the traI structural gene and application of purified mutant TraI proteins for in vitro assays served to evaluate the functional importance of conserved amino acid residues. Two regions of TraI designated as motifs I and III are involved in catalyzing the cleaving-joining reaction. Motif I carries the tyrosine residue (Tyr-22), which covalently attaches TraI in a transesterification reaction to the 5' terminus of the cleaved DNA. Motif III contains one histidine residue (His-116) essential for relaxase activity and therefore proposed to activate the aromatic hydroxyl group of tyrosine 22 by proton abstraction. Exchange of a serine residue (Ser-74), located in motif II, against alanine prevents formation of stable relaxosomes but strongly enhances topoisomerase activity of the combination TraI/TraJ on form I oriT DNA. Motif II therefore might represent the DNA recognition domain of TraI. Our studies allowed us to establish a model of the interplay of three motifs located in the N-terminal region (amino acid positions 19-124) of TraI.
- Subjects :
- DNA, Bacterial
Exonucleases
Protein Conformation
Molecular Sequence Data
Biology
Relaxase
Biochemistry
Conserved sequence
Substrate Specificity
chemistry.chemical_compound
Plasmid
Escherichia coli
Amino Acid Sequence
Cloning, Molecular
Site-directed mutagenesis
Molecular Biology
Peptide sequence
Conserved Sequence
Binding Sites
Base Sequence
Sequence Homology, Amino Acid
Bacterial conjugation
Escherichia coli Proteins
DNA Helicases
Cell Biology
Relaxosome
Recombinant Proteins
Models, Structural
chemistry
Oligodeoxyribonucleotides
Genes, Bacterial
Conjugation, Genetic
Mutagenesis, Site-Directed
Tyrosine
DNA
Plasmids
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 269
- Issue :
- 4
- Database :
- OpenAIRE
- Journal :
- The Journal of biological chemistry
- Accession number :
- edsair.doi.dedup.....a9e3d28d699e43b905a712d91d281adf