Restriction-Modification Systems

Recent studies with Type I R-M Systems:

Identifying the recognition sequences of Type I R-M enzymes

(Ryu & Rowsell, 2008)

Although DNA-recognition sequences are among the most important characteristics of restriction enzymes and their corresponding methylases, determination of the recognition sequence of a Type-I restriction enzyme is a complicated procedure. To facilitate this process we have previously developed plasmid R-M tests and the computer program RM search. To specifically identify Type-I isoschizomers, we engineered a pUC19 derivative plasmid, pTypeI, which contains all of the 27 Type-I recognition sequences in a 248-bp DNA fragment. Furthermore, a series of 27 plasmids (designated reference plasmids'), each containing a unique Type-I recognition sequence, were also constructed using pMECA, a derivative of pUC vectors. In this study, we tried those vectors on 108 clinical E. coli strains and found that 48 strains produced isoschizomers of Type I enzymes. A detailed study of 26 strains using these reference plasmids' revealed that they produce seven different isoschizomers of the prototypes: EcoAI, EcoBI, EcoKI, Eco377I, Eco646I, Eco777I and Eco826I. One strain EC1344 produces two Type I enzymes (EcoKI and Eco377I).

Motifs in EcoR124I HsdR

(Sisakova et al., 2008)

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.

Modeling the structure of the HsdR subunit of EcoR124I

(Obarska-Kosinska et al., 2008)

Type I restriction-modification (RM) systems are large, multifunctional enzymes composed of three different subunits. HsdS and HsdM form a complex in which HsdS recognizes the target DNA sequence, and HsdM carries out methylation of adenosine residues. The HsdR subunit, when associated with the HsdS-HsdM complex, translocates DNA in an ATP-dependent process and cleaves unmethylated DNA at a distance of several thousand base-pairs from the recognition site. The molecular mechanism by which these enzymes translocate the DNA is not fully understood, in part because of the absence of crystal structures. To date, crystal structures have been determined for the individual HsdS and HsdM subunits and models have been built for the HsdM-HsdS complex with the DNA. However, no structure is available for the HsdR subunit. In this work, the gene coding for the HsdR subunit of EcoR124I was re-sequenced, which showed that there was an error in the published sequence. This changed the position of the stop codon and altered the last 17 amino acid residues of the protein sequence. An improved purification procedure was developed to enable HsdR to be purified efficiently for biophysical and structural analysis. Analytical ultracentrifugation shows that HsdR is monomeric in solution, and the frictional ratio of 1.21 indicates that the subunit is globular and fairly compact. Small angle neutron-scattering of the HsdR subunit indicates a radius of gyration of 3.4 nm and a maximum dimension of 10 nm. We constructed a model of the HsdR using protein fold-recognition and homology modelling to model individual domains, and small-angle neutron scattering data as restraints to combine them into a single molecule. The model reveals an ellipsoidal shape of the enzymatic core comprising the N-terminal and central domains, and suggests conformational heterogeneity of the C-terminal region implicated in binding of HsdR to the HsdS-HsdM complex.

A history of the plasmid R124

(Youell and Firman, 2008)

For a number of years I have believed that I should write a history of our (and others) research on the plasmid R124 and the R-M system it encodes.  At long last I have managed to do this and also to introduce some of the new single molecule work I have been involved with and explain, briefly, how we hope to make commercial use of the EcoR124I molecular motor.

Phosphorylation of the motor subunit of EcoKI

(Cajthamlová et al., 2007)

Representative enzymes from the three main families of Type R-M Systems were analysed using immunoblotting in vivo and a sensitive phosphorylation assay in vitro and the HsdR (motor) subunit of the Type IA enzyme EcoKI was found to be the only subunit that was phosphorylated. Of particular interest was the observation that this phosphorylation only occurs in vivo when the subunit is co-produced with HsdM and HsdS, indicating that the phosphorylation is in someway linked to endonuclease activity.  It is interesting to ask whether this phosphorylation activity is in anyway connected with Restriction Alleviation and ClpXP proteolysis of HsdR.

Recent Publications

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Copyright © 2006 Keith Firman
Last modified: 23-Jul-2008