T4 Endonuclease IV

An improved procedure for the purification of T4 endonuclease IV from T4+-infected Escherichia coli cells is described. The enzyme preparations are highly concentrated, free of exonuclease contamination, and display about 200-fold greater activity toward single stranded DNA than toward duplex DNA. The limit product obtained by exhaustive digestion of single stranded DNA with endonuclease IV is 25% acid-soluble and consists of oligonucleotides with an average chain length of about 50 nucleotides. The enzyme may be useful in sequencing DNA. A second endonuclease which exhibits a preference for denatured DNA was also purified from T4+-infected E. coli cells and was named T4 endonuclease III. This enzyme is clearly distinguishable from T4 endonuclease IV on the basis of its chromatographic behavior. Furthermore, endonuclease III is able to act on denatured T4 DNA, whereas endonuclease IV does not; endonuclease III does not produce appreciable acid-soluble material from polydeoxycytidylate, whereas endonuclease IV does; and endonuclease III produces oligonucleotides whose 5′-terminal mononucleotides are not specific, whereas the oligonucleotides produced by endonuclease IV end exclusively in 5′-dCMP.