[25] Purification and Properties of a Pyrimidine Dimer-Specific Endonuclease from E. Coli Infected with Bacteriophage T4

The chapter explains purification and properties of a pyrimidine dimer-specific endonuclease from E. coli infected with bacteriophage T4. The pyrimidine dimer-specific endonuclease of bacteriophage T4 is coded by the v (denV) gene. The enzyme has been called endonuclease V of phage T4 or the T4 uv endonuclease. For detecting pyrimidine dimers a preparation of enzyme free of activity against unirradiated DNA or DNA containing other types of damage is required. The chapter describes several methods for assaying T4 endonuclease V as well as the partial purification and general properties of the enzyme. The chapter discusses a reproducible procedure for obtaining stable preparations of the enzyme which can be used as a specific probe for pyrimidine dimers. For the purpose of purification, the enzyme can be assayed by one of three general methods— namely, coupled nuclease assay, filter binding assay, and DNA nicking assay. The chapter further discusses enzyme purification.