Bacteriophage T7 endonuclease. I. Properties of the enzyme purified from T7 phage-infected Escherichia coli B.

An endonuclease activity present in extracts from Escherichia coli B infected with T7f phage has been purified 2500fold. This enzyme, T7 endonuclease I, is the product of gene 3, and its activity is maximal 7.5 min after infection at 37”. The activity is at least 100 times greater with single stranded DNA as substrate than with duplex DNA. The limit product obtained by digestion of single stranded DNA with the endonuclease is about 50% acid soluble and consists of oligonucleotides with a chain length of about 10. The enzyme produces single strand and double strand breaks in duplex DNA, and the limit product obtained when double stranded DNA is digested with the endonuclease consists of fragments of duplex DNA whose strands are about 125 nucleotides long. T7 endonuclease I produces breaks with 5’-phosphate and 3’-hydroxyl termini. Although all four deoxynucleoside monophosphates are found at the 5’-terminus, the enzyme makes fragments with predominantly pyrimidine-containing nucleotides at their 5’-termini. The enzyme acts equally well on E. coli DNA and T4, T7, and X phage DNA.