Ultraviolet-induced DNA Damage Stimulates Topoisomerase

An antibody-based method was used to examine genomic DNA cleavage by endogenous topoisomerases in living cells. The method quantifies cleavable (covalent) complex formation in vivo after exposure to topoisomerase poisons, as reported previously (D. Subramanian et al., Cancer Res., 55: 2097-2103, 1995). Unexpectedly, exposing cells to UVB irradiation stim ulated endogenous topoisomerase I-DNA covalent complex formation by as much as 8-fold, even in the absence of drugs that stabilize the cleavable complex. Covalent complexes are not a result of nonspecific UV proteinDNA cross-linking; rather, they result from the enzymatic activity of topoisomerase I on genomic DNA. Because the action of topoisomerase II on genomic DNA was not affected by UVB exposure, the observation appears to be specific for type I. Topoisomerase I is rapidly mobilized onto the genome (within 12 min after UVB exposure); however, topoisomerase I polypeptide levels did not show a corresponding increase, suggesting that preexisting enzyme is being recruited to sites of DNA damage. Complexes persist up to 5 h post-UV exposure (concurrent with the period of active DNA repair), and their formation is independent of S phase. These findings can be partially explained by the fact that in vitro topoisomerase I activity on UV-damaged DNA tends to favor formation of cleavage complexes; thus, a higher yield of covalent complexes are detected at or near cyclopyrimidine dimer lesions. Because repair-deficient cells are additionally compromised in their ability to recruit topoisomerase I, a direct role for the enzyme in DNA excision repair process in vivo is proposed that may be related to the activity of the xeroderma pigmentosiini complementation group D helicase. Finally, these results collectively demonstrate that topoisomerase I is a repair-proficient topoisomerase in vivo. lowing UV irradiation (6). These results are consistent with the idea that DPC formation after UV irradiation represents some form of repair process or cellular response to DNA damage. Because topoi somerases represent a class of nuclear proteins that covalently bind genomic DNA, their relationship with DPC formation has been ex amined. Topoisomerases are enzymes that alter the topology of DNA by breaking and resealing DNA. The catalytic cycle of the type I enzyme involves the following steps: DNA binding. DNA breakage, passage of the DNA through the enzyme-DNA gate, resealing DNA breaks, and release of the enzyme from the binding site (for reviews see Refs. 8-10). Topoisomerase activity has been implicated in DNA templating processes, such as transcription (11-14), replication (15, 16), and, possibly, DNA repair (17-20). During the breakage step, an interme diate, which consists of a covalent complex between the broken DNA and the active site tyrosine, is formed (21-23). Both topoisomerase I and topoisomerase II form covalent bonds with DNA (concurrent with DNA breaks); therefore, either of these proteins may be responsible for the observed DPC formation following UVB irradiation. To examine the involvement of topoisomerases in UVB-induced DPCs, we used an in vivo assay (the ICT bioassay) to measure topoisomerase activity on genomic DNA in intact cells following UVB irradiation (24, 25). The results demonstrate that UVB specifi cally stimulates the formation of covalent complexes between topoi somerase I and DNA covalent complexes but not topoisomerase II-DNA covalent complexes.