ABSTRACT
An Escherichia coli RFL47 DNA fragment containing the Eco47IR and Eco47II restriction-modification (R-M) system has been cloned and sequenced. A clone carrying this system has been selected by its ability to restrict phage lambda in vivo. The sequence of 5360 bp was determined, and its analysis revealed three major open reading frames (ORF) corresponding to two restriction endonucleases (ENases) and one DNA methyltransferase (MTase): R.Eco47II (239 amino acid (aa)), R.Eco47I (230 aa) and M.Eco47II (417 aa). The M.Eco47II aa sequence possesses all conserved domains typical for m5C MTases and its variable region has a high homology with M.Sau96I and M.SinI. The ORF harboring a predicted helix-turn-helix motif upstream from the eco47IR gene has been found. No sequence resembling the eco47IM gene has been detected in the complete fragment sequenced, although disrupted ORF, possibly corresponding to the transposase-encoding gene, has been found in the intergenic area between eco47IIM and eco47IR. No homology was found between the ENases; however, both revealed homology with their isoschizomers, R.SinI and R.Sau96I.
Subject(s)
DNA-Cytosine Methylases/biosynthesis , Deoxyribonucleases, Type II Site-Specific/biosynthesis , Escherichia coli Proteins , Escherichia coli/enzymology , Genes, Bacterial , Amino Acid Sequence , Bacteriophage lambda/metabolism , Base Sequence , Cloning, Molecular/methods , DNA-Cytosine Methylases/genetics , DNA-Cytosine Methylases/metabolism , Deoxyribonucleases, Type II Site-Specific/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Escherichia coli/genetics , Molecular Sequence Data , Open Reading Frames , Restriction Mapping , Sequence Homology, Amino AcidABSTRACT
The complete type-II Cfr9I restriction-modification (R-M) system of Citrobacter freundii strain RFL9, recognizing the DNA sequence CCCGGG, has been cloned and expressed, and functionally active enzymes have been produced in Escherichia coli. Both the methyltransferase (MTase; M.Cfr9I) and restriction endonuclease (ENase; R.Cfr9I) were found to be encoded on a 2.3-kb cloned fragment in the same transcriptional orientation, but differing in translational phases. The last codon (underlined) (ATGA) of the MTase-encoding gene (Cfr9IM) overlaps with the start codon for the ENase-encoding gene (overlined) (cfr9IR). A nucleotide sequence complementary to a predicted Shine-Dalgarno sequence preceding cfr9IR is within this gene. Predicted free energy (delta G) for formation of the mRNA secondary structure involving these complementary sequences was found to be -16.1 kcal/mol. Amino-acid sequence homology of 80% was found between R.Cfr9I and R.XcyI.
Subject(s)
Citrobacter freundii/enzymology , DNA Modification Methylases/genetics , Deoxyribonucleases, Type II Site-Specific/genetics , Protein Biosynthesis/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Citrobacter freundii/genetics , Cloning, Molecular , DNA, Bacterial/isolation & purification , Molecular Sequence Data , Nucleic Acid Conformation , Oligodeoxyribonucleotides , Reading Frames , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
The specificity of three DNA methyltransferases M.Alw26I, M.Eco31I and M.Esp3I, isolated from Acinetobacter Iwoffi RFL26, Escherichia coli RFL31 and Hafnia alvei RFL3+, respectively, was determined. All the enzymes methylate both strands of asymmetric recognition sites yielding m5C in the top-strand and m6A in the bottom-strand, as below: 5'-GTm5CTC 5'-GGTm5CTC 5'-CGTm5CTC 3'-Cm6AGAG 3'-CCm6AGAG 3'-GCm6AGAG (M.Alw26I) (M.Eco31I) (M.Esp3I) They are the first members of type IIs methyltransferases that modify different types of nucleotides in the recognition sequence.
Subject(s)
Acinetobacter/enzymology , DNA Modification Methylases/metabolism , DNA-Cytosine Methylases/metabolism , Enterobacteriaceae/enzymology , Escherichia coli/enzymology , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Base Sequence , Kinetics , Molecular Sequence Data , Oligodeoxyribonucleotides/metabolism , Substrate SpecificityABSTRACT
The sequences coding for DNA[cytosine-N4]methyltransferases MvaI (from Micrococcus varians RFL19) and Cfr9I (from Citrobacter freundii RFL9) have been determined. The predicted methylases are proteins of 454 and 300 amino acids, respectively. Primary structure comparison of M.Cfr9I and another m4C-forming methylase, M.Pvu II, revealed extended regions of homology. The sequence comparison of the three DNA[cytosine-N4]-methylases using originally developed software revealed two conserved patterns, DPF-GSGT and TSPPY, which were found similar also to those of adenine and DNA[cytosine-C5]-methylases. These data provided a basis for global alignment and classification of DNA-methylase sequences. Structural considerations led us to suggest that the first region could be the binding site of AdoMet, while the second is thought to be directly involved in the modification of the exocyclic amino group.
