Primary sequence of the EcoRII endonuclease and properties of its fusions with beta-galactosidase.
The EcoRII endonuclease cleaves DNA containing the sequence CC(A/T)GG before the first cytosine. The methylation of the second cytosine in the sequence by either the EcoRII methylase or Dcm, a chromosomally coded protein in Escherichia coli, inhibits the cleavage. The gene for the EcoRII endonuclease was mapped by analysis of derivatives containing linker insertions, transposon insertions, and restriction fragment deletions. Surprisingly, plasmids carrying the wild-type endonuclease gene and the EcoRII methylase gene interrupted by transposon insertions appeared to be lethal to dcm+ strains of E. coli. We conclude that not all the EcoRII/Dcm recognition sites in the cellular DNA are methylated in dcm+ strains. The DNA sequence of a 1650-base pair fragment containing the endonuclease gene was determined. It revealed an open reading frame that could code for a 45.6-kDa protein. This predicted size is consistent with the known size of the endonuclease monomer (44 kDa). The endonuclease and methylase genes appear to be transcribed convergently from separate promoters. The reading frame of the endonuclease gene was confirmed at three points by generating random protein fusions between the endonuclease and beta-galactosidase, followed by an analysis of the sequence at the junctions. One of these fusions is missing 18 COOH-terminal amino acids of the endonuclease but still displays significant ability to restrict incoming phage in addition to beta-galactosidase activity. No striking similarity between the sequence of the endonuclease and any other protein in the PIR data base was found. The knowledge of the primary sequence of the endonuclease and the availability of the various constructs involving its gene should be helpful in the study of the interaction of the enzyme with its substrate DNA.
Bhagwat
A S
AS
Cold Spring Harbor Laboratory, New York 11724.
Johnson
B
B
Weule
K
K
Roberts
R J
RJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005696:Galactosidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D015183:Restriction Mapping; D001616:beta-Galactosidase / Q000378:metabolism*
C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D005696:Galactosidases; D001616:beta-Galactosidase