DNA determinants of restriction. Bacteriophage T4 endonuclease II-dependent cleavage of plasmid DNA in vivo.
Endonuclease II of coliphage T4 is necessary for the in vivo restriction of plasmid DNA in phage-infected cells. Double-stranded restriction cleavage at 12 sites in pBR322 commenced before 10-min postinfection with T4 at 37 degrees C and proceeded more slowly in the presence of competing phage DNA than in its absence, utilizing the same sites in both cases; in a 200-base pair segment of the plasmid, single-stranded nicks also were frequent. The plasmid sites were cleaved with a speed that varied with the site, yielding frequencies of cleavage at different sites varying between 10 and 90%, at 50-min postinfection. All sites contained good matches to a consensus, 5'-GRCCGCNTYGC-3', most frequently cleaved around the variable central base pair, generating fragments with blunt ends or 1-2-base 5' overhangs. Using the frequency of cleavage to determine a weighted consensus, a larger sequence, 5'-CGRCCGCNTTGSYNGC-3', was identified. Thus, DNA sequence elements 3' to the cut site appear important for rapid cleavage. Several models describing the sequence-dependent structure of DNA suggest structural anomalies around the cleavage sites. The endonuclease II restriction system is most similar to type II systems, although it differs from known type II systems in several respects.
Department of Microbiology, University of Uppsala Biomedical Center, Sweden.
K
K
Carlson
M
M
Krabbe
A C
AC
Nyström
L D
LD
Kosturko
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D015139:Blotting, Southern; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids
D004269:DNA, Bacterial; D004279:DNA, Viral; D004262:DNA Restriction Enzymes