Human DNA Topoisomerase I-mediated Cleavages Stimulated by Ultraviolet Light-induced DNA Damage

Abstract

DNA topoisomerases have been proposed as the proteins involved in the formation of the DNA-protein cross-links detected after ultraviolet light (UV) irradiation of cellular DNA. This possibility has been investigated by studying the effects of UV-induced DNA damage on human DNA topoisomerase I action. UV lesions impaired the enzyme's ability to relax negatively supercoiled DNA. Decreased relaxation activity correlated with the stimulation of cleavable complexes. Accumulation of cleavable complexes resulted from blockage of the rejoining step of the cleavage-religation reaction. Mapping of cleavage sites on the pAT153 genome indicated UV-induced cleavage at discrete positions corresponding to sites stimulated also by the topoisomerase I inhibitor camptothecin, except for one. Subsequent analysis at nucleotide level within the sequence encompassing the UV-specific cleavage site revealed the precise positions of sites stimulated by camptothecin with respect to those specific for UV irradiation. Interestingly, one of the UV-stimulated cleavage sites was formed within a sequence that did not contain dimerized pyrimidines, suggesting transmission of the distortion, caused by photodamage to DNA, into the neighboring sequences. These results support the proposal that DNA structural alterations induced by UV lesions can be sufficient stimulus to induce cross-linking of topoisomerase I to cellular DNA.Cyclobutane pyrimidine dimers (CPDs) 1 and (6 -4) photoproducts are the most prevalent lesions produced in DNA by ultraviolet (UV) light (1). However, pyrimidine dimers are not the only photochemical effect of UV light on cellular DNA. It has been shown that UV radiation induces also the formation of DNA-protein cross-links (2-4). Proteinase K treatment abolishes the cross-linking effect and reveals the presence of cryptic DNA strand breaks. Since this cross-linking is partially repaired, it has been suggested that this non-dimer DNA damage may play an important role in the biological effect of UV radiation (2, 4). DNA topoisomerases have been proposed as possible candidates for the protein(s) involved in UV-induced DNAprotein cross-linking (4). This proposal is consistent with the formation of transient single and double strand breaks during DNA topoisomerase reactions, with covalent attachment of the enzymes to one terminus of the DNA nick (reviewed in Ref. 5).DNA topoisomerases are ubiquitous enzymes involved in a number of crucial cellular processes, including replication, transcription, and recombination. A relationship between cellular responses to DNA damage and topoisomerases has been proposed (6 -9). The catalytic cycle of DNA topoisomerases can be divided into several steps: 1) enzyme-DNA binding; 2) DNA cleavage, resulting in a covalent attachment between the protein and one terminus of the DNA nick; 3) DNA strand passage; 4) poststrand passage DNA religation concerted with the enzyme turnover (reviewed in Ref. UV photoproducts cause alterations of the DNA conformation that can affect the activity of DNA processin...

Trapping of Mammalian Topoisomerase I and Recombinations Induced by Damaged DNA Containing Nicks or Gaps

“…Thus, our data further demonstrate that top1 can be trapped by DNA damage. Recently, trapping of top1 by DNA mismatches, abasic sites (21), and UV damage (38) has also been reported. At the present time, it is not known whether such a trapping is part of DNA repair (39) or cell death pathways.…”

Gadd45, a p53-Responsive Stress Protein, Modifies DNA Accessibility on Damaged Chromatin

“…For example, damage produced by shortwave UV radiation interfered with the proper catalytic activity of Top1 (40), resulting in a stimulation of cleavage complexes in the absence of a Top1 poison. The UV-stimulated Top1 cleavage sites are apparently in close proximity to cyclopyrimidine clusters (64).…”

Tyrosyl-DNA Phosphodiesterase as a Target for Anticancer Therapy

“…In a similar manner, a number of naturally occurring DNA lesions, such as strand breaks [15], abasic sites, base mismatches, and certain oxidized or modified bases [16], have also been shown in vitro to physically block the Top1 religation reaction through the misalignment the 5′-hydroxyl with the tyrosyl-DNA phosphodiester backbone. In cells, both UV irradiation- [17] and hydrogen peroxide-induced [18] DNA damage have also been shown to trap Top1 cleavage complexes. Repair of these irreversible Top1-DNA cleavage complexes has been suggested as an important part of DNA metabolism given that mutants defective in specific DNA repair pathways demonstrate increased sensitivity to Top1 inhibitors.…”