MUTATIONAL SPECIFICITY OF OXIDATIVE DNA DAMAGE

In this paper we describe our studies on the mutagenic consequences of oxidative DNA damage introduced by radiation-induced OH radicals (OH) and by exposure to singlet oxygen (O-1(2)), released by thermo-dissociation of the endoperoxide 3,3'-(1,4-naphthalidene) dipropionate (NDPO2). We have made use of M13mp10 bacteriophage and pUC18 plasmid DNA, containing a 144 base pair (bp) insert in the lacZ alpha gene. This 144 bp insert was used as a mutational target sequence. When dilute aqueous solutions of double-stranded (ds) M13mp10 (plus 144 bp insert) were gamma-irradiated in the presence of oxygen (O2; 100% OH) or nitrous oxide (N2O; 90% OH, 10% H), very specific mutation spectra were found. Mainly bp substitutions were observed, of which C/G to G/C transversions are the predominant type. Moreover, the mutations are for the most part concentrated into two mutational hot spots: a minor and major one. Differences between the oxic (O2) and anoxic (N2O) mutation spectra could also be observed. Under N2O - 1 bp deletions were detected, which are absent in the presence of O2, and in the anoxic spectrum more C/G to A/T transversions are present. To investigate whether these differences were due to the small amount of H radicals, which are formed under N2O, ds M13mp10 (plus 144 bp insert) was exposed to gamma-rays in phosphate buffer under nitrogen (55% H, 45% OH). Under these conditions a remarkable shift was observed from C/G --> G/C to C/G --> A/T transversions, while the mutations were far more scattered along the 144 bp sequence and no - 1 bp deletions were detected. These results strongly suggest that H radicals do not cause - 1 bp deletions, but may be responsible for the observed C/G to A/T transversions. The kind of bp substitution not only appeared to be dependent on the type of the water radicals, but also appeared to be strongly influenced by the replicon in which the target sequence is incorporated. When an oxygenated solution of pUC18 plasmid DNA (plus 144 bp insert) is irradiated, mainly C/G to A/T transversions were found at the same major hot spot instead of C/G to G/C transversions when the 144 hp sequence is part of M13mp10 DNA. Finally, in agreement with the observation that 102 reacts preferentially with guanine in DNA, a guanine is involved in most of the mutations scored after exposure of single-stranded (ss) M13mp10 DNA to NDPO2-generated O-1(2). A preference for G to T transversions appeared to exist, while a relatively larger number of mutations were characterized by the fact that a G was deleted. Again the mutations tend to accumulate in the same major hot spot found for the OH-induced mutations. From our data together with those obtained by others, it can be concluded that oxidative DNA damage, introduced by direct reaction of OH radicals or 102 with DNA, lead preferentially to mutations on G/C base pairs, while the chance that a mutation is formed and the type of mutation is strongly dependent on the sequence in which a G/C base pair is present and on the mode of replication of the DNA molecule.