INTERACTION OF SINGLET OXYGEN WITH DNA AND BIOLOGICAL CONSEQUENCES

To study the interaction of singlet oxygen (O-1(2)) with DNA and the biological consequences of O-1(2)-induced DNA damage, we used the thermodissociable endoperoxide of 3,3'-(1,4 naphthalidene) dipropionate (NDPO2) as a generator of free O-1(2) in reactions with (1) 2'-deoxynucleoside 3'-monophosphates (dNps), (2) an oligonucleotide (16-mer) having one deoxyguanine (dG), (3) native and denaturated rat kidney DNA and (4) single-stranded (ss) and double-stranded (ds) bacteriophage M13mp10 DNA. Using both anion exchange and reversed phase HPLC and P-32-postlabeling analyses, it was found that exposure of the various dNps to chemically generated O-1(2) led to a detectable reaction with dGp and not with dAp, dCp, d5mCp or Tp. The reaction with dGp led to degradation of this nucleotide and the formation of a large number of reaction products, one of which could be identified as 7-hydro-8-oxo-2'-deoxyguanosine 3'-monophosphate (8-oxo-dGp). A second product could tentatively be identified as a formamido pyrimidine derivative of dGp (Fapy-dGp). When ss DNA, ds DNA or the oligonucleotide were exposed to O-1(2), the formation of 8-oxo-dG could also be demonstrated. With the oligonucleotide, we found a so far unidentified reaction product. Under the same reaction conditions the yield of 8-oxo-dG was about 8-fold higher in ss DNA than in ds DNA. In ss DNA 8-oxo-dG seemed to be a more prominent product than in the case of reaction of O-1(2) with free dGp. Reaction of O-1(2) with ss or ds M13mp10 DNA led to biological inactivation of these DNAs, ss DNA being at least 100-fold more sensitive than ds DNA. It could be concluded that inactivation of the ss DNA must be largely due to O-1(2)-induced DNA lesions other than 8-oxo-dG. In agreement with the observed preferential reaction of O-1(2) with dG most of the so far sequenced mutations, induced by O-1(2) in a 144 bp mutation target sequence inserted in the lacZalpha gene of ss or ds M13mp10 DNA, occurred at a G or G/C base pair respectively. A preference for G(C) to T(A) transversions can be observed for which 8-oxo-dG might have been responsible. In ss DNA a significant number of the mutations are characterized by the fact that a G is deleted.