Action of the single-stranded DNA specific nuclease S 1 on double-stranded DNA

Abstract

The single-stranded DNA specific nuclease S 1 (Ando, T. (1966) Biochim. Biophys. Acta 114, 158–168) puts single-stranded breaks into double-stranded ΦX174 and T7 DNAs. The enzyme first converts the supercoiled ΦX174 RF I ★ ★ The double-stranded replicative form of Φ174 DNA is abbreviated throughout this paper as RF I (closed supercoiled form) or RF II (open circular form). into the relaxed form RF II, and then puts an increasing number of nicks into both of the DNA strands of the duplex. These breaks can only be observed upon denaturation of the molecule. This double-stranded DNA nicking activity of S 1 nuclease is low compared with the single-stranded DNA digestion activity, and during the time it takes to completely digest ΦX174 single-stranded DNA to acid soluble nucleotides ( i.e. approx. 5000 phosphodiester bond cleavages), only two to three nicks are put into the double-stranded ΦX174 DNA molecule ( i.e. four to six phosphodiester bond cleavages). As the enzyme preparation used gave a single band on a sodium dodecyl sulphate polyacrylamide gel, it is tentatively concluded that the S 1 nuclease not only digests single-stranded DNA to nucleotides, but also puts single-strand breaks in double-stranded DNA.