Enzymatic Repair of DNA, 1. 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 Vol. 63; no. 1; pp. 144 - 151
• Main Authors: Kaplan, Joan C., Kushner, Sidney R., Grossman, Lawrence
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01-05-1969
• Subjects: Biochemistry; Centrifugation; Chemical suspensions; Deoxyribonucleases - isolation & purification; Dimers; DNA; DNA - radiation effects; Enzymes; Glycols; Irradiation; Magnesium; Micrococcus - enzymology; Nucleotides; Potassium phosphates; Radiation Effects; Thymine; Tritium; Ultraviolet Rays
• 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.