ENZYMATIC REPAIR OF DNA, I. PURIFICATION OF TWO ENZYMES INVOLVED IN THE EXCISION OF THYMINE DIMERS FROM ULTRAVIOLET-IRRADIATED DNA

• Published in: Proceedings of the National Academy of Sciences - PNAS 1969-05, Vol.63 (1), p.144-151
• Main Authors: Kaplan, Joan C., Kushner, Sidney R., Grossman, Lawrence
• Format: Article
• Language: English
• Subjects: Biological Sciences: Biochemistry
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.63.1.144

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.