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PROJECT #1 |
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Primary reference: Subramanian, D., Rosenstein, B. and Muller, M.T. (1998) Ultraviolet-induced DNA damage stimulates topoisomerase I-DNA complex fromation in vivo: Possible relationship with DNA repair. Cancer Research 58:976-984 Related paper(s) in press or submitted
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ABSTRACT. 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 (Subramanian et al., Cancer Research 55:2097-2103, 1995). Unexpectedly, exposing cells to UVB-irradiation stimulates 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 protein/DNA crosslinking but result from the enzymatic activity of topoisomerase I on genomic DNA. Since topoisomerase II action on genomic DNA was not affected by UVB exposure, the observation appears to be specific for type I topoisomerases. Topoisomerase I is rapidly mobilized onto the genome (within 12 minutes 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 hours 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. Since 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 XPD helicase. Finally, these results collectively demonstrate that type I is a repair proficient topoisomerase in vivo. |
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