A EUKARYOTIC GENE ENCODING AN ENDONUCLEASE THAT SPECIFICALLY REPAIRS DNA DAMAGED BY ULTRAVIOLET-LIGHT

Many eukaryotic organisms, including humans, remove ultraviolet (UV) d amage from their genomes by the nucleotide excision repair pathway, wh ich requires more than 10 separate protein factors. However, no nucleo tide excision repair pathway has been found in the filamentous fungus Neurospora crassa. We have isolated a new eukaryotic DNA repair gene f rom N.crassa by its ability to complement UV-sensitive Escherichia col i cells. The gene is altered in a N.crassa mus-18 mutant and responsib le for the exclusive sensitivity to UV of the mutant. Introduction of the wildtype mus-18 gene complements not only the mus-18 DNA repair de fect of N.crassa, but also confers UV-resistance on various DNA repair -deficient mutants of Saccharomyces cerevisiae and a human xeroderma p igmentosum cell line. The cDNA encodes a protein of 74 kDa with no seq uence similarity to other known repair enzymes. Recombinant mus-18 pro tein was purified from E.coli and found to be an endonuclease for UV-i rradiated DNA. Both cyclobutane pyrimidine dimers and (6-4)photoproduc ts are cleaved at the sites immediately 5' to the damaged dipyrimidine s in a magnesium-dependent, ATP-independent reaction. This mechanism, requiring a single polypeptide designated UV-induced dimer endonucleas e for incision, is a substitute for the role of nucleotide excision re pair of UV damage in N.crassa.