PHOTOREVERSAL-DEPENDENT RELEASE OF THYMIDINE AND THYMIDINE MONOPHOSPHATE FROM PYRIMIDINE DIMER-CONTAINING DNA EXCISION FRAGMENTS ISOLATED FROM ULTRAVIOLET-DAMAGED HUMAN-FIBROBLASTS

To elucidate the enzymatic excision-repair process operative on cyclobutane-type pyrimidine photodimers in human dermal fibroblasts, we have examined excised dimer-containing material recovered in the trichloroacetic acid soluble fraction from far-ultraviolet-irradiated (254 nm, 40 J m-2) and incubated (24 h) cell cultures. The excised DNA photoproducts were found in oligonucleotide fragments with an estimated mean chain length of .apprx. 3.7 bases. Exposure of these isolated excision fragments, labeled with [3H]thymidine (dT), to a secondary, dimer-photoreversing fluence of far-UV (5.5 kJ m-2) resulted in the release of free dT and thymidine monophosphate (TMP). Photorelease of these two radioactive species was measured by high-performance liquid chromatography, with TMP being detected as the increase in dT following bacterial alkaline phosphatase treatment. These data imply that the photoliberated dT and TMP moieties were attached to the excision fragments solely by the cyclobutane ring of the dimer. No evidence was obtained for the photoliberation of free thymine, thus corroborating a conclusion reached by others that the excision of dimers in human cells is not initiated by scission of an intradimer N-glycosyl bond. The sum of the tritium label recovered in dT plus TMP corresponded to approximately 40% of that disappearing from thymine-containing dimers on photoreversal, suggesting that in about 80% of the isolated excision fragments the dimer is located at one end of the oligonucleotide and contains a break in its internal phosphodiester bond. These findings, coupled with our recent studies on xeroderma pigmentosum complementation group A and D cells [Paterson, M. C., Gentner, N.E., Middlestadt, M. V., and Weinfeld, M. (1984) J. Cell. Physiol., Suppl. 3, 45-62], lead us to propose cleavage of the intradimer phosphodiester linkage as the initial reaction in the multistep excision-repair process acting on pyrimidine dimers in the DNA of normal human cells.