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Human DNA topoisomerase I-mediated cleavages stimulated by ultraviolet light-induced DNA damage
A Lanza1, S Tornaletti, C Rodolfo
1Istituto di Genetica Biochimica ed Evoluzionistica del CNR, Via Abbiategrasso, 207-27100 Pavia, Italy.
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.
Insights
Ultraviolet light (UV) damage to DNA impairs DNA topoisomerase I activity, leading to DNA-protein cross-links. This study shows UV lesions directly stimulate topoisomerase I to form these cross-links.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA topoisomerases are crucial for managing DNA topology.
- DNA-protein cross-links are observed after UV irradiation.
- The role of DNA topoisomerases in UV-induced DNA-protein cross-linking is unclear.
Purpose of the Study:
- To investigate the involvement of human DNA topoisomerase I in forming DNA-protein cross-links after UV irradiation.
- To characterize the effects of UV-induced DNA damage on DNA topoisomerase I activity.
Main Methods:
- Assessing DNA relaxation activity of human DNA topoisomerase I on supercoiled DNA.
- Mapping UV-induced and camptothecin-stimulated cleavage sites.
- Nucleotide-level analysis of cleavage sites.
Main Results:
- UV lesions significantly reduced DNA topoisomerase I relaxation activity.
- UV damage stimulated the formation of cleavable complexes, indicating a blockage in the rejoining step.
- Specific UV-induced cleavage sites were identified, with one occurring outside a typical pyrimidine dimer site, suggesting distortion transmission.
Conclusions:
- DNA topoisomerase I is implicated in UV-induced DNA-protein cross-link formation.
- UV-induced DNA structural alterations can directly trigger topoisomerase I cross-linking.
- The findings provide mechanistic insight into DNA repair and photodamage response.