Increased protein flexibility leads to promiscuous protein--DNA interactions in type IC restriction-modification systems.
We have investigated the role of a four amino acid element that is repeated twice and three times, respectively, in the specificity polypeptides of the two allelic restriction-modification systems EcoR124 and EcoR124/3. We had earlier shown that this difference in amino acid sequence between the two systems is solely responsible for the different DNA sequence specificities of the two systems. The effect of single amino acid substitutions and small insertion and deletion mutations on restriction activity and modification specificity was determined in vivo by phage infection assays and in vitro by methylation of DNA with purified modification methylases. Mutant restriction-modification systems with changes in the number and the length of the central amino acid repeats exhibited decreased restriction activity and in some cases relaxed substrate specificity. Our data strongly support the idea that the repetitive amino acid motif in the specificity polypeptides forms part of a flexible interdomain linker. It may be responsible for positioning on the DNA the two major specificity polypeptide domains which are thought to contact independently the half sites of the split recognition sequences typical for all type I restriction-modification systems.
Gubler
M
M
Microbiology Department, Biozentrum, University of Basel, Switzerland.
Bickle
T A
TA
D000483:Alleles; D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics*; D015254:DNA Modification Methylases / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011485:Protein Binding; D015183:Restriction Mapping; D013379:Substrate Specificity
D004247:DNA; D015254:DNA Modification Methylases; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI