Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains.

• Keywords
• DNA restriction enzymes, Domain interactions, Molecular mechanics, Molecular modeling, Multisubunit enzyme complex, Principal components analysis,
• MeSH
• Adenosine Triphosphate chemistry   MeSH
• Enzyme Activation MeSH
• Principal Component Analysis MeSH
• DNA Helicases chemistry   genetics   metabolism   MeSH
• Hydrolysis MeSH
• Protein Interaction Domains and Motifs * MeSH
• Protein Conformation MeSH
• Multienzyme Complexes chemistry   MeSH
• Mutation MeSH
• Protein Subunits chemistry   genetics   metabolism   MeSH
• Deoxyribonucleases, Type I Site-Specific chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Molecular Dynamics Simulation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• DNA Helicases MeSH
• Multienzyme Complexes MeSH
• Protein Subunits MeSH
• Deoxyribonucleases, Type I Site-Specific MeSH

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

• Keywords
• DNA restriction enzymes, Domain interactions, E. coli, Molecular modeling, Multisubunit enzyme complex,
• Publication type
• Journal Article MeSH

Restriction-modification systems protect bacteria from foreign DNA. Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA-cleavage and ATP-dependent DNA translocation activities located on endonuclease/motor subunit HsdR. The recent structure of the first intact motor subunit of the type I restriction enzyme from plasmid EcoR124I suggested a mechanism by which stalled translocation triggers DNA cleavage via a lysine residue on the endonuclease domain that contacts ATP bound between the two helicase domains. In the present work, molecular dynamics simulations are used to explore this proposal. Molecular dynamics simulations suggest that the Lys-ATP contact alternates with a contact with a nearby loop housing the conserved QxxxY motif that had been implicated in DNA cleavage. This model is tested here using in vivo and in vitro experiments. The results indicate how local interactions are transduced to domain motions within the endonuclease/motor subunit.

• MeSH
• Adenosine Triphosphate chemistry   metabolism   MeSH
• Amino Acid Motifs MeSH
• DNA chemistry   metabolism   MeSH
• Phenotype MeSH
• Genotype MeSH
• Hydrolysis MeSH
• Catalysis MeSH
• Kinetics MeSH
• Conserved Sequence MeSH
• Quantum Theory MeSH
• Lysine MeSH
• Mutation MeSH
• Mutagenesis, Site-Directed MeSH
• Deoxyribonucleases, Type I Site-Specific chemistry   genetics   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• DNA MeSH
• endodeoxyribonuclease EcoR124I MeSH Browser
• Lysine MeSH
• Deoxyribonucleases, Type I Site-Specific MeSH

This review summarizes the main results obtained in the fields of general and molecular microbiology and microbial genetics at the Institute of Microbiology of the Academy of Sciences of the Czech Republic (AS CR) [formerly Czechoslovak Academy of Sciences (CAS)] over more than 50 years. Contribution of the founder of the Institute, academician Ivan Málek, to the introduction of these topics into the scientific program of the Institute of Microbiology and to further development of these studies is also included.

• MeSH
• Academies and Institutes history   MeSH
• History, 20th Century MeSH
• Genetics, Microbial history   MeSH
• Molecular Biology history   MeSH
• Check Tag
• History, 20th Century MeSH
• Publication type
• Journal Article MeSH
• Historical Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Geographicals
• Czech Republic MeSH

BACKGROUND: Type I restriction-modification (R-M) systems are the most complex restriction enzymes discovered to date. Recent years have witnessed a renaissance of interest in R-M enzymes Type I. The massive ongoing sequencing programmes leading to discovery of, so far, more than 1 000 putative enzymes in a broad range of microorganisms including pathogenic bacteria, revealed that these enzymes are widely represented in nature. The aim of this study was characterisation of a putative R-M system EcoA0ORF42P identified in the commensal Escherichia coli A0 34/86 (O83: K24: H31) strain, which is efficiently used at Czech paediatric clinics for prophylaxis and treatment of nosocomial infections and diarrhoea of preterm and newborn infants. RESULTS: We have characterised a restriction-modification system EcoA0ORF42P of the commensal Escherichia coli strain A0 34/86 (O83: K24: H31). This system, designated as EcoAO83I, is a new functional member of the Type IB family, whose specificity differs from those of known Type IB enzymes, as was demonstrated by an immunological cross-reactivity and a complementation assay. Using the plasmid transformation method and the RM search computer program, we identified the DNA recognition sequence of the EcoAO83I as GGA(8N)ATGC. In consistence with the amino acids alignment data, the 3' TRD component of the recognition sequence is identical to the sequence recognized by the EcoEI enzyme. The A-T (modified adenine) distance is identical to that in the EcoAI and EcoEI recognition sites, which also indicates that this system is a Type IB member. Interestingly, the recognition sequence we determined here is identical to the previously reported prototype sequence for Eco377I and its isoschizomers. CONCLUSION: Putative restriction-modification system EcoA0ORF42P in the commensal Escherichia coli strain A0 34/86 (O83: K24: H31) was found to be a member of the Type IB family and was designated as EcoAO83I. Combination of the classical biochemical and bacterial genetics approaches with comparative genomics might contribute effectively to further classification of many other putative Type-I enzymes, especially in clinical samples.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• DNA Restriction-Modification Enzymes genetics   metabolism   MeSH
• Escherichia coli enzymology   genetics   MeSH
• Genomics MeSH
• Escherichia coli Proteins genetics   metabolism   MeSH
• Antibodies, Bacterial metabolism   MeSH
• Deoxyribonucleases, Type I Site-Specific genetics   metabolism   MeSH
• Base Sequence MeSH
• Sequence Homology, Nucleic Acid MeSH
• Sequence Alignment MeSH
• Genetic Complementation Test MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• DNA Restriction-Modification Enzymes MeSH
• HsdM protein, Bacteria MeSH Browser
• HsdR protein, E coli MeSH Browser
• Escherichia coli Proteins MeSH
• Antibodies, Bacterial MeSH
• Deoxyribonucleases, Type I Site-Specific MeSH