Most cited article - PubMed ID 17620716
Purification, crystallization and preliminary X-ray analysis of the HsdR subunit of the EcoR124I endonuclease from Escherichia coli
Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.
- MeSH
- Adenosine Triphosphate chemistry metabolism MeSH
- DNA, Bacterial MeSH
- Escherichia coli genetics metabolism MeSH
- Exodeoxyribonuclease V chemistry genetics metabolism MeSH
- Gene Expression MeSH
- Nucleic Acid Conformation MeSH
- Crystallography, X-Ray MeSH
- Models, Molecular MeSH
- Mutation MeSH
- Plasmids chemistry metabolism MeSH
- Protein Subunits chemistry genetics metabolism MeSH
- Protein Sorting Signals MeSH
- Escherichia coli Proteins chemistry genetics metabolism MeSH
- Deoxyribonucleases, Type I Site-Specific chemistry genetics metabolism MeSH
- Signal Transduction MeSH
- DNA Cleavage MeSH
- Protein Structure, Tertiary MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Adenosine Triphosphate MeSH
- DNA, Bacterial MeSH
- exodeoxyribonuclease V, E coli MeSH Browser
- Exodeoxyribonuclease V MeSH
- HsdR protein, E coli MeSH Browser
- Protein Subunits MeSH
- Protein Sorting Signals MeSH
- Escherichia coli Proteins MeSH
- Deoxyribonucleases, Type I Site-Specific MeSH
