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.

Institute of Microbiology v v i Academy of Sciences of the Czech Republic Prague Czech Republic

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A residue of motif III positions the helicase domains of motor subunit HsdR in restriction-modification enzyme EcoR124I

The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

Functional coupling of duplex translocation to DNA cleavage in a type I restriction enzyme

Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I

General and molecular microbiology and microbial genetics in the IM CAS

The interrelationship of helicase and nuclease domains during DNA translocation by the molecular motor EcoR124I