Tris-dependent site-specific cleavage of Streptomyces lividans DNA

Abstract

During conventional gel electrophoresis, Streptomyces lividans DNA undergoes site-specific double-strand cleavage at the positions of closely opposed unstable modifications introduced into the DNA in vivo. We investigated this electrophoretic instability and demonstrated that it was dependent on Tris. Tris buffer was activated at the anode to generate a nucleolytic species; prior to activation, Tris was not able to cleave the DNA. The nucleolytic species was shown to react with thiourea, which could thus protect the DNA from strand cleavage. Non-degradative electrophoresis of the DNA could also be achieved in an alternative buffer such as Hepes.