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Characterization of Excision Repair in Neurospora crassa

^a Institute of Radiation Biology, University of Tennessee, Knoxville, Tennessee
^b Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

ABSTRACT

The excision of pyrimidine dimers from the deoxyribonucleic acid (DNA) of Neurospora crassa was examined. Postirradiation incubation in the presence of several chemicals known to inhibit various repair systems indicated that caffeine reduced the rate of excision twofold, but did not inhibit excision completely as did proflavine and quinacrine. Examination of the time course of excision showed that repair occurs at a relatively rapid rate: approximately 60 dimers excised per min after 500 ergs/mm². Further evidence for rapid excision was obtained by sedimentation analysis of DNA; the maximal number of breaks introduced during repair was three, suggesting that breaks are repaired almost as fast as they are made and that only a few dimers are repaired at a time. Repair synthesis was measured by prelabeling the DNA with ¹⁵N and D₂O, and then subjecting the DNA to equilibrium density gradient centrifugation after postirradiation incubation with ³²P. Accumulation of single-strand breaks with increasing dose of ultraviolet radiation suggested that the limiting step was subsequent to the incision and excision steps of repair. Equilibrium CsCl centrifugation demonstrated that the limiting step in excision was repair synthesis.

¹ Present address: Division of Rhaumatic and Genetic Diseases, Department of Medicine, Duke University Medical Center, Durham, N.C. 27706.