Unpaired bases in phage DNA after gamma-irradiation in-situ and in-vitro

Phage Lambda DNA, gamma-irradiated in-situ and in-vitro, has been analyzed for unpaired bases by melting, reannealing, and cleavage with Sl nuclease which is specific for single-stranded DNA. DNA, irradiated in-situ, i.e., in the phage particle, contained sites being sensitive to Sl nuclease. These single-stranded lesions were passed over and conserved during reannealing, whereas adjacent DNA regions reannealed specifically. Complementary base-pairing was restored after Sl nuclease treatment. Comparison of the Tm-data before and after Sl nuclease treatment indicated that the single-stranded regions were removed by the enzyme. In contrast, DNA irradiated in-vitro, i.e., gamma-irradiated in aqueous solution, failed to match complementarily and was not sensitive to Sl nuclease. Thus it appears that lesions leading to unpaired bases were randomly distributed in DNA irradiated in-vitro, but occurred in clusters after irradiation in-situ. Most probably these clusters contain damaged bases which in turn caused localized disruption of the hydrogen bonds between complementary base pairs.