A Novel Model of Enzymatic Repair of UV-Induced DNA Damage in Human Cells
We have obtained evidence for the existence of a pre-incision step in the excision-repair pathway operating on UV-induced cyclobutyl pyrimidine dimers in cultured human cells. Demonstration of the occurrence of this reaction includes the following five observations. 1) In the majority (85%) of the dimercontaining excision fragments isolated from post-UV incubated normal fibroblasts, the intradimer phosphodiester linkage is cleaved, but the cleavage products remain joined together by the cyclobutane ring of the dimer. 2) Various human specimens (e.g., lymphocytes and liver tissue) contain an activity, termed intracyclobutyl pyrimidine dimer-DNA phosphodiesterase (IDP), capable of mediating this intradimer backbone-nicking reaction; the IDP activity is optimally expressed at pH 5.5 and is associated with a Mg+ +-stimulated, 52 kDa protein, which has now been purified 3500-fold from human liver. 3) In the genomic DNA extracted from post-UV incubated xeroderma pigmentosum (XP) complementation group D cells, which are totally defective in dimer excision, a fraction (~15%) of the dimer sites have a hydrolysed internal phosphodiester bond. 4) These backbone-nicked dimers tend to accumulate in transcriptionally active (i.e., c-myc) genomic sequences in UV-damaged XP D cells. 5) Such modified dimer sites also appear transiently in normal fibroblasts at early times after UV treatment. Together, these findings suggest that the intradimer backbone cleavage reaction, perhaps by reducing local conformational stress, may promote transcription on a UV-damaged template and thereby defer the actual excision-repair event to a later time.
A novel nuclease digestion/HPLC assay has also been developed to characterize further human excision-repair pathways(s) active on two major classes of UV lesions, cyclobutyl dimers and (6-4) photoproducts. By analyzing isolated UV lesions which accumulate in excision fragments during incubation of UV-treated cells, the excision kinetics for these two classes of lesions have been shown to differ greatly. (6-4) photoproducts are removed in their entirety by 6 hr, whereas cyclobutyl dimers are processed much more slowly, with a retention half-life of ~16 hr. Unlike the situation for dimers noted above, (6-4) photoproducts are released with the internal phosphodiester linkage intact. Finally, fibroblast strains from XP groups A, C, D, and E exhibit a deficiency in the repair of cyclobutyl dimers and/or (6-4) photoproducts which is unique for each group, signifying that mutations at different loci underlie the different malfunctional repair patterns.
KeywordsXeroderma Pigmentosum Phosphodiester Bond Pyrimidine Dimer Phosphodiester Linkage Snake Venom Phosphodiesterase
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