Nucleic Acids Research Advance Access published online on October 6, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp790
Nucleic Acid Enzymes |
DNA cleavage and methylation specificity of the single polypeptide restriction–modification enzyme LlaGI
1DNA-Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK and 2Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
*To whom correspondence should be addressed. Tel: +44 117 331 2158; Fax: +44 117 331 2168; Email: mark.szczelkun{at}bristol.ac.uk
Received July 28, 2009. Revised September 4, 2009. Accepted September 8, 2009.
LlaGI is a single polypeptide restriction–modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a 
-family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5'-CTnGAyG-3' (where n is A, G, C or T and y is C or T). Methylation of the recognition site occurred on only one strand (the non-degenerate dA residue of 5'-CrTCnAG-3' being methylated at the N6 position). Double strand DNA breaks at distant, random sites were only observed when two head-to-head oriented, unmethylated copies of the site were present; single sites or pairs in tail-to-tail or head-to-tail repeat only supported a DNA nicking activity. dsDNA nuclease activity was dependent upon the presence of ATP or dATP. Our results are consistent with a directional long-range communication mechanism that is necessitated by the partial site methylation. In the accompanying manuscript [Smith et al. (2009) The single polypeptide restriction–modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops], we demonstrate that this communication is via 1-dimensional DNA loop translocation. On the basis of this data and that in the third accompanying manuscript [Smith et al. (2009) An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI], we propose that LlaGI is the prototype of a new sub-classification of Restriction-Modification enzymes, named Type I SP (for Single Polypeptide).
Present address: Jytte Josephsen, Øresund Food Network, Nørre Voldgade 16, DK 1358, Copenhagen K, Denmark.