Exposure to ultraviolet radiation, which leads to the formation of mutagenic and cytotoxic DNA lesions such as cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs), can be potentially fatal. The way UVA forms DNA lesions and alters DNA topology and mechanics is still unclear, unlike the cases of UVC and UVB. Herein, Atomic Force Microscopy (AFM) and AFM-based Force Spectroscopy (AFS) have been employed to investigate the topological and mechanical properties of single DNA molecules, bare or E. coli cell-encapsulated, with or without UVA (solar or from UV lamp) treatment. It is observed that both the dsDNA transitions, i.e., 'B' to stretched 'S' conformation and melting transition, are lost in UVA dose-dependent manner. Presumably, this is due to formation of the CPDs and 6-4 lesions that form inter-strand cross-links, causing dsDNA strand separation difficult. Gradual reduction in DNA extension length upon prolonged treatment with UVA-only radiation or sunlight (where, 95 % of solar UV is UVA) also indicates formation of the inter-strand cross-links, since such cross-links can reduce DNA flexibility and increase DNA stiffness. Although these observations are common for both bare and cell-encapsulated DNA, the UVA dose at which the distinctive reversible B-S and melting transition faded away varied widely from 240 kJ/m2 (bare DNA) to 900 kJ/m2 (cellular DNA). The UV-induced DNA damage was also evident in observation of increased number of open circular and linearized topologies, as formed due to single-strand and double-strand breaks, respectively, at damage sites, upon combined action of the apurinic/apyrimidinic site-specific endonucleases IV and V. The extent of DNA damage was further quantified by enzyme-linked immunosorbent assay, which is found to be correlated to the single molecule information.
Keywords: 6–4 PPs; AFM-based force spectroscopy; CPDs; Cellular DNA damage; Single molecule; Sunlight; UVA radiation.
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