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

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

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

The type I restriction-modification enzyme EcoR124I comprises three subunits with the stoichiometry HsdR2/HsdM2/HsdS1. The HsdR subunits are archetypical examples of the fusion between nuclease and helicase domains into a single polypeptide, a linkage that is found in a great many other DNA processing enzymes. To explore the interrelationship between these physically linked domains, we examined the DNA translocation properties of EcoR124I complexes in which the HsdR subunits had been mutated in the RecB-like nuclease motif II or III. We found that nuclease mutations can have multiple effects on DNA translocation despite being distinct from the helicase domain. In addition to reductions in DNA cleavage activity, we also observed decreased translocation and ATPase rates, different enzyme populations with different characteristic translocation rates, a tendency to stall during initiation and altered HsdR turnover dynamics. The significance of these observations to our understanding of domain interactions in molecular machines is discussed.

• MeSH
• Adenosine Triphosphatases metabolism   MeSH
• Amino Acid Motifs MeSH
• Biological Transport MeSH
• Biological Assay MeSH
• DNA Helicases chemistry   MeSH
• DNA metabolism   MeSH
• Endonucleases chemistry   MeSH
• Escherichia coli enzymology   MeSH
• Kinetics MeSH
• Molecular Motor Proteins chemistry   metabolism   MeSH
• Molecular Sequence Data MeSH
• Mutagenesis MeSH
• Mutant Proteins chemistry   metabolism   MeSH
• Optical Tweezers MeSH
• Protein Subunits chemistry   metabolism   MeSH
• Deoxyribonucleases, Type I Site-Specific chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Structure, Tertiary MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• DNA Helicases MeSH
• DNA MeSH
• endodeoxyribonuclease EcoR124I MeSH Browser
• Endonucleases MeSH
• Molecular Motor Proteins MeSH
• Mutant Proteins MeSH
• Protein Subunits MeSH
• Deoxyribonucleases, Type I Site-Specific MeSH