Enzymatic repair of DNA, 1. Purification of two enzymes involved in the excision of thymine dimers from ultraviolet-irradiated DNA

Abstract

Two nucleases that catalyze the excision of photoproducts from UV-irradiated DNA have been extensively purified from M. luteus (M. lysodeikticus). The first enzyme, an endonuclease, has been purified 5000-fold and is free of conflicting nuclease activities. It introduces single-strand breaks into irradiated DNA but does not act on native or single-stranded DNA. The purified enzyme is activated but not dependent on Mg(++) and has an approximate molecular weight of 15,000. Photoproduct excision is absolutely dependent on the second enzyme, a magnesium requiring exonuclease. This enzyme, which has been purified 1000-fold, is devoid of conflicting nucleases. It hydrolyzes irradiated and unirradiated denatured DNA at the same rate, but has no activity on RNA. It only acts on double-stranded DNA which has been both irradiated and pretreated with the endonuclease. The combined action of the endo- and exonuclease results in the quantitative removal of photoproduct regions from UV-irradiated DNA. Approximately five nucleotides are released for every single-strand incision.