Topoisomerase I recruitment in human cells following DNA damage

Exposure of human cells to the UVB wavelengths causes induction of low levels of DNA/protein crosslinks which increase in number upon incubation. These crosslinks are always accompanied by the appearance of single-strand DNA breaks and probably represent some form of repair process or cellular response to DNA damage. Here, we show that type I topoisomerase (topo) is one of the proteins participating in the formation of the DNA/protein crosslinks and breaks. These findings are based upon an in vivo assay that measures endogenous topo activity on genomic DNA using antibodies to assess covalent complexes formed between the enzyme and DNA. The results reveal that UVB irradiation stimulates topo/DNA complex formation by as much as 7 fold in human cells. Formation of crosslinks takes place rapidly as adduce are detectable within 12 min. post-irradiation and persist for at least 15 hours; however, over this period, topo polypeptide levels remain constant. These data suggest that topo is recruited by cellular processes associated with DNA repair or damage recognition. A role for topo in DNA repair is further demonstrated by using repair deficient cell lines to measure UVB induced topo/DNA crosslinks. The results show that cells derived from the xeroderma pigmentosum complementation group A exhibited similar elevation in topo activity as the parental cell line; however, xeroderma pigmentosum complementation group D were deficient in the formation of topo/DNA crosslinks. Additionally, topo binds preferentially to UV damaged DNA over normal B-DNA in vitro. Although foe exact role of topo in DNA repair is not known, recognition of lesions or repair patches or recruitment of the enzyme into repair sites may be related to this increased activity. Finally, camptothecin, a topo inhibitor, further stimulated adduct formation in UVB irradiated cells beyond levels observed in cell treated with either UVB or camptothecin alone. Mechanistically, this could be exploited to enhance tumor killing using combinatorial chemotherapy. Initially, DNA damaging agents could be used to recruit topo to repair sites. This could be followed with camptothecin to increase topo mediated complex formation at these repair sites resulting in greater cell death.