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
• adenosintrifosfatasy metabolismus   MeSH
• aminokyselinové motivy MeSH
• biologický transport MeSH
• biotest MeSH
• DNA-helikasy chemie   MeSH
• DNA metabolismus   MeSH
• endonukleasy chemie   MeSH
• Escherichia coli enzymologie   MeSH
• kinetika MeSH
• molekulární motory chemie   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutageneze MeSH
• mutantní proteiny chemie   metabolismus   MeSH
• optická pinzeta MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• restrikční endonukleasy typu I chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• DNA-helikasy MeSH
• DNA MeSH
• endodeoxyribonuclease EcoR124I MeSH Prohlížeč
• endonukleasy MeSH
• molekulární motory MeSH
• mutantní proteiny MeSH
• podjednotky proteinů MeSH
• restrikční endonukleasy typu I 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
• bakteriální proteiny genetika   metabolismus   MeSH
• DNA restrikčně-modifikační enzymy genetika   metabolismus   MeSH
• Escherichia coli enzymologie   genetika   MeSH
• genomika MeSH
• proteiny z Escherichia coli genetika   metabolismus   MeSH
• protilátky bakteriální metabolismus   MeSH
• restrikční endonukleasy typu I genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie nukleových kyselin MeSH
• sekvenční seřazení MeSH
• testy genetické komplementace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA restrikčně-modifikační enzymy MeSH
• HsdM protein, Bacteria MeSH Prohlížeč
• HsdR protein, E coli MeSH Prohlížeč
• proteiny z Escherichia coli MeSH
• protilátky bakteriální MeSH
• restrikční endonukleasy typu I MeSH

Type I restriction-modification (R-M) endonucleases are composed of three subunits--HsdR, required for restriction, and HsdM and HsdS which can produce a separate DNA methyltransferase. The HsdS subunit is required for DNA recognition. In this paper we describe the effect of cloned EcoKI and EcoR124I hsd genes on the resulting R-M phenotype. The variability in the expression of the wild type (wt) restriction phenotype after cloning of the wt hsd genes in a multicopy plasmid in Escherichia coli recA+ background suggests that the increased production of the restriction endonuclease from pBR322 is detrimental to the cell and this leads to the deletion of the cloned hsd genes from the hybrid plasmid and/or inactivation of the enzyme. The effect of a mutation in E. coli recA gene on the expression of R-M phenotype is described and discussed in relation to the role of the cell surface and the localization of the restriction endonuclease in the cell.

• MeSH
• bakteriální geny MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• DNA restrikčně-modifikační enzymy genetika   metabolismus   MeSH
• Escherichia coli enzymologie   genetika   růst a vývoj   MeSH
• klonování DNA MeSH
• mutace MeSH
• plazmidy genetika   MeSH
• proteiny z Escherichia coli * MeSH
• RecA-rekombinasy genetika   MeSH
• regulace genové exprese u bakterií * MeSH
• restrikční endonukleasy typu I genetika   metabolismus   MeSH
• restrikční enzymy genetika   metabolismus   MeSH
• síran hořečnatý farmakologie   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA restrikčně-modifikační enzymy MeSH
• endodeoxyribonuclease EcoKI MeSH Prohlížeč
• endodeoxyribonuclease EcoR124I MeSH Prohlížeč
• HsdR protein, E coli MeSH Prohlížeč
• HSDS protein, Bacteria MeSH Prohlížeč
• proteiny z Escherichia coli * MeSH
• RecA-rekombinasy MeSH
• restrikční endonukleasy typu I MeSH
• restrikční enzymy MeSH
• síran hořečnatý MeSH