Interaction of singlet oxygen with DNA and biological consequences

Abstract

To study the interaction of singlet oxygen (¹O₂) with DNA and the biological consequences of ¹O₂-induced DNA damage, we used the thermodissociable endoperoxide of 3,3′-(1,4 naphtalidene) dipropionate (NDPO₂) as a generator of free ¹O₂ 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 ³²P-postlabeling analyses, it was found that exposure of the various dNps to chemically generated ¹O₂ 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 ¹O₂, the formation of 8-oxo-dG could also be demonstrated. With the oligonucleotide, we found a so far unidentifed 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 ¹O₂ with free dGp.

Reaction of ¹O₂ 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 ¹O₂-induced DNA lesions other than 8-oxo-dG. In agreement with the observed preferential reaction of ¹O₂ with dG most of the so far sequenced mutations, induced by ¹O₂ in a 144 bp mutation target sequence inserted in the lacZα gene of ss or ds M13mp10 DNA, occurred at a