Nucleic Acids Research, 1992, Vol. 20, No. 22 6051-6056
© 1992
ENZYMOLOGY |
Cloning and sequence analysis of the genes coding for Eco57l type IV restriction-modification enzymes
Institute of Biotechnology FERMENTAS Graiciuno 8, 2028 Vilnius, Lithuania
* To whom correspondence should be addressed
Received July 6, 1992. Revised October 26, 1992. Accepted October 26, 1992.
A 6.3 kb fragment of E.coli RFL57 DNA coding for the type IV restriction-modification system Eco571 was cloned and expressed inE.coli RR1. A 5775 bp region of the cloned fragment was sequenced which contains three open reading frames (ORF). The methylase gene is 1623 bp long, corresponding to a protein of 543 amino acids (62 kDa); the endonuclease gene is 2991 bp in length (997 amino acids, 117 kDa). The two genes are transcribed convergently from different strands with their 3'-ends separated by 69 bp. The third short open reading frame (186 bp, 62 amino acids) has been identified, that precedes and overlaps by 7 nucleotides the ORF encoding the methylase. Comparison of the deduced Eco571 endonuclease and methylase amino acid sequences revealed three regions of significant similarity. Two of them resemble the conserved sequence motifs characteristic of the DNA[adenine-N6] methylases. The third one shares similarity with corresponding regions of the PaeR7l, Taql, Cv/BIII, Pstl, BamHI and Hincll methylases. Homologs of this sequence are also found within the sequences of the PaeR7l, Pstt and BamHI restriction endonucleases. This is the first example of a family of cognate restriction endonucleases and methylases sharing homologous regions. Analysis of the structural relationship suggests that the type IV enzymes represent an intermediate in the evolutionary pathway between the type III and type II enzymes.
+ Present address: Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, NY 11724, USA
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