Nucleic Acids Research Advance Access published online on September 30, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp795
Nucleic acid Enzymes |
An Mrr-family nuclease motif in 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, Faculty of Life Sciences, University of Copenhagen, 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 9, 2009.
Bioinformatic analysis of the putative nuclease domain of the single polypeptide restriction–modification enzyme LlaGI reveals amino acid motifs characteristic of the Escherichia coli methylated DNA-specific Mrr endonuclease. Using mutagenesis, we examined the role of the conserved residues in both DNA translocation and cleavage. Mutations in those residues predicted to play a role in DNA hydrolysis produced enzymes that could translocate on DNA but were either unable to cleave the polynucleotide track or had reduced nuclease activity. Cleavage by LlaGI is not targeted to methylated DNA, suggesting that the conserved motifs in the Mrr domain are a conventional sub-family of the PD-(D/E)XK superfamily of DNA nucleases.
Present address: Jytte Josephsen, Øresund Food Network, Nørre Voldgade 16, DK 1358, Copenhagen K, Denmark.