Factors influencing resistance of UV-irradiated DNA to the restriction endonuclease cleavage

Photochemical probing of the B–A conformational transition in a linearized pUC19 DNA and its polylinker region

“…We showed in our recent studies that restriction endonucleases can be used to detect conformation dependent UV damage in DNA [35] and that this approach can be used to map the B-A transition along linearized pUC19 DNA [36]. Here we extend our previous results with primer extension studies of the damage caused in DNA by UV light in the course of B-A transition.…”

“…We were able to detect the B-A transition with 16 tested restriction enzymes, because the A-form DNA was much more resistant to UV light damage than its B-form [33,35]. As the changes in cleavability with restriction endonucleases reflect UV lightinduced damage within the whole recognition sequences (at least 6 bp) plus flanking nucleotides, we wanted to analyze the damage in more detail.…”

“…In fact, it can also be a monofunctional photoproduct of an isolated pyrimidine base. We focused our attention mainly on the polylinker of pUC19 plasmid, because this 57 bp long DNA segment is a continual row of recognition sequences of ten restriction endonucleases and because it was exploited in our previous studies [35,36].…”

“…Sequencing dideoxy reactions were performed according to the manufacturer's instructions (Amersham Life Sciences). Radioactive DNA fragments were observed by autoradiography at − 70°C with an intensifier screens and quantified by laser densitometry of autoradiograms [35]. Baselines were calculated from the graphs, peak integrals (baseline subtracted) were calculated and used for analysis.…”

“…They detected the damage by chemical sequencing methods to probe the B-A transition of a part of the 5S ribosomal RNA gene. Recently we have extended this approach by using restriction endonucleases to detect the UV light damage of DNA in the B-form and A-form [35]. We have furthermore shown that the UV damage connected with its detection by restriction endonucleases can be used to detect B-A transition along the linearized pUC19 plasmid DNA [36].…”