ULTRAVIOLET-INDUCED DNA-DAMAGE STIMULATES TOPOISOMERASE-I DNA COMPLEX-FORMATION IN-VIVO - POSSIBLE RELATIONSHIP WIT DNA-REPAIR

An antibody-based method was used to examine genomic DNA cleavage by e ndogenous topoisomerases in living cells, The method quantifies cleava ble (covalent) complex formation in vivo after exposure to topoisomera se poisons, as reported previously (D. Subramanian et al., Cancer Res. , 55: 2097-2103, 1995), Unexpectedly, exposing cells tit UVB irradiati on stimulated endogenous topoisomerase I-DNA covalent complex formatio n by as much as 8-fold, even in the absence of drugs that stabilize th e cleavable complex, Covalent complexes are not a result of nonspecifi c UV protein-DNA cross-linking; rather, they result from the enzymatic activity of topoisomerase I on genomic DNA, Because the action of top oisomerase II on genomic DNA was not affected by UVB exposure, the obs ervation 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 incr ease, suggesting that preexisting enzyme is being recruited to sites o f DNA damage, Complexes persist up to 5 h post-UV exposure (concurrent with the period of active DNA repair), and their formation is indepen dent of S phase. These findings can be partially explained try the fac t that in vitro topoisomerase I activity on UV-damaged DNA tends to fa vor formation of cleavage complexes; thus, a higher yield of covalent complexes are detected at or near cyclopyrimidine dimer lesions, Becau se repair-deficient cells are additionally compromised in their abilit y to recruit topoisomerase I, a direct role for the enzyme in DNA exci sion repair process in vivo is proposed that may be related to the act ivity of the xeroderma pigmentosum complementation group D helicase, F inally, these results collectively demonstrate that topoisomerase I is a repair-proficient topoisomerase in vivo.