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
• adenosintrifosfát chemie   metabolismus   MeSH
• DNA bakterií MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exodeoxyribonukleasa V chemie   genetika   metabolismus   MeSH
• exprese genu MeSH
• konformace nukleové kyseliny MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• mutace MeSH
• plazmidy chemie   metabolismus   MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• proteiny - lokalizační signály MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• restrikční endonukleasy typu I chemie   genetika   metabolismus   MeSH
• signální transdukce MeSH
• štěpení DNA MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Type I restriction-modification enzyme EcoR124I is an ATP-dependent endonuclease that uses dsDNA translocation to locate and cleave distant non-specific DNA sites. Bioinformatic analysis of the HsdR subunits of EcoR124I and related Type I enzymes showed that in addition to the principal PD-(E/D)xK Motifs, I, II and III, a QxxxY motif is also present that is characteristic of RecB-family nucleases. The QxxxY motif resides immediately C-terminal to Motif III within a region of predicted alpha-helix. Using mutagenesis, we examined the role of the Q and Y residues in DNA binding, translocation and cleavage. Roles for the QxxxY motif in coordinating the catalytic residues or in stabilizing the nuclease domain on the DNA are discussed.

• MeSH
• aminokyselinové motivy MeSH
• DNA metabolismus   MeSH
• exodeoxyribonukleasa V chemie   MeSH
• financování organizované MeSH
• kinetika MeSH
• molekulární sekvence - údaje MeSH
• mutageneze MeSH
• podjednotky proteinů chemie   MeSH
• restrikční endonukleasy typu I genetika   chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• substituce aminokyselin MeSH
• transport proteinů MeSH