Mutational analysis and homology-based modeling of the IntDOT core-binding domain
| Autor: | Jeffrey F. Gardner, Abigail A. Salyers, Joel P. Cioni, Brian M. Swalla, Karolina Malanowska |
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| Rok vydání: | 2009 |
| Předmět: |
Models
Molecular DNA Mutational Analysis Molecular Sequence Data Biology Microbiology Catalysis chemistry.chemical_compound Viral Proteins Recombinase Escherichia coli Bacteroides Amino Acid Sequence Tyrosine Molecular Biology Genetics chemistry.chemical_classification Integrases Active site Bacteriophage lambda Enzymes and Proteins Integrase Amino acid Protein Structure Tertiary chemistry Biochemistry Attachment Sites Microbiological Mutation biology.protein Sequence Alignment DNA Binding domain |
| Zdroj: | Journal of bacteriology. 191(7) |
| ISSN: | 1098-5530 |
| Popis: | Tyrosine recombinases mediate a wide range of important genetic rearrangement reactions. Models for tyrosine recombinases have been based largely on work done on the integrase of phage lambda and recombinases like Cre, Flp, and XerC/D. All of these recombinases share a common amino acid signature that is important for catalysis. Several conjugative transposons (CTns) encode recombinases that are also members of the tyrosine recombinase family, but the reaction that they catalyze differs in that recombination does not require homology in the attachment sites. In this study, we examine the role of the core-binding (CB) domain of the CTnDOT integrase (IntDOT) that is located adjacent to the catalytic domain of the protein. Since there is no crystal structure for any of the CTn integrases, we began with a predicted three-dimensional structure produced by homology-based modeling. Amino acid substitutions were made at positions predicted by the model to be close to the DNA. Mutant proteins were tested for the ability to mediate integration in vivo and for in vitro DNA-binding, cleavage, and ligation activities. We identified for the first time nonconserved amino acid residues in the CB domain that are important for catalytic activity. Mutant proteins with substitutions at three positions in the CB domain are defective for DNA cleavage but still proficient in ligation. The positions of the residues in the complex suggest that the mutant residues affect the positioning of the cleaved phosphodiester bond in the active site without disruption of the ligation step. |
| Databáze: | OpenAIRE |
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