PRODUCTION OF BREAKS IN SINGLE-STRANDED AND DOUBLE-STRANDED FORMS OF BACTERIOPHAGE-PHI-X174 DNA BY PROFLAVINE AND LIGHT TREATMENT

Irradiation at 440 + 360 nm and a fluence rate of 3.8 kJm-2 min-1, of both complexes previously formed between proflavine and either .vphi.X circular single-stranded (ss) DNA or .vphi.X supercoiled duplex (RFI)DNA, induces single-strand scissions in the 2 DNA under consideration. Linear .vphi.X ssDNA molecules are detected by sedimentation through alkaline sucrose gradients. After treatment of the .vphi.X RF [replicative form] I DNA, the degree of degradation is the same whether it is measured under neutral or alkaline conditions, indicating that alkaline-labile bonds are not created; double-strand breaks can only be detected after accumulation of single-strand breaks. In addition to the amount of proflavine bound to the DNA and the duration of irradiation, the following factors influence the nicking activity of the treatment: the DNA structure (the .vphi.X RFI DNA is much more sensitive than the .vphi.X ssDNA); the ionic strength of the medium during irradiation (a high value of 0.5 leads to a markedly increased efficiency); the addition of cysteamine (this latter compound decreases the reaction rate); and the irradiation wavelength (after irradiation at 440 nm alone, the reaction occurs at a reduced rate and is sensitive to NaN3). The kinetics of the nicking reaction does not follow a single-hit curve, showing that at least 1 primary lesion occurs prior to strand breakage. Strand scission cannot be detected after irradiation of the proflavine-DNA complexes at the low fluence rate, causing a decrease in the infectivity of both .vphi.X ss and .vphi.X RFI DNA. Similarly, the sedimentation pattern of the DNA extracted from treated .vphi.X 174 phages 99.9% inactivated, is identical to that of the control ssDNA, although more drastic treatments are susceptible to induce single strand breaks inside the phage head. The unknown lesion(s) that is biologically important does not prevent the treated DNA from penetrating into the host [Escherichia coli] cells.