S1 nuclease does not cleave DNA at single-base mis-matches

Abstract

Three assays have been designed to detect the cleavage of duplex ΦX174 DNA at single-base mis-matches. Studies with S₁ nuclease failed to detect cleavage at mis-matches. S₁ nuclease digestion at 37 and 55°C failed to produce a preferential degradation of a multiply mis-matched heteroduplex when compared to a mis-match-free homoduplex as analyzed by sedimentation on sucrose gradients. Other heteroduplex templates were not cleaved by S₁ nuclease at a defined single-base mis-match when assayed by gel electrophoresis or by marker rescue. In all cases, the amount of S₁ nuclease employed was at least 10-times more than that required to render a single-stranded ΦX174 DNA molecule completely acid soluble. The rate of hydrolysis of single-base mis-matches by S₁ nuclease was estimated to be less than 0.016% of the rate at a base in single-strand ΦX174 DNA. In no instance did we detect activity by S₁ nuclease directed at mis-matched sites in our template molecules. Similarly, the single-strand specific endonuclease from Neurospora crassa does not cleave heteroduplex templates at a defined single-base mismatch when assayed by marker rescue.