8-hydroxyguanine and 5-6-dihydrothymine

Clustered DNA damage induced by a single radiation track is a unique feature of ionizing radiation. Using a plasmid-based assay in Escherichia coli, we previously found significantly higher mutation frequencies for bistranded clusters containing 7,8-dihydro-8-oxoguanine (8-oxoG) and 5,6-dihydrothymine (DHT) than for either a single 8-oxoG or a single DHT in wild type and in glycosylase-deficient strains of E. coli. This indicates that the removal of an 8-oxoG from a clustered damage site is most likely retarded compared to the removal of a single 8-oxoG. To gain further insights into the processing of bistranded base lesions, several potential repair intermediates following 8-oxoG removal were assessed. Clusters, such as DHT+apurinic/apyrimidinic (AP) and DHT+GAP have relatively low mutation frequencies, whereas clusters, such as AP+AP or GAP+AP, significantly reduce the number of transformed colonies, most probably through formation of a lethal double strand break (DSB). Bistranded AP sites placed 3' to each other with various interlesion distances also blocked replication. These results suggest that bistranded base lesions, i.e., single base lesions on each strand, but not clusters containing only AP sites and strand breaks, are repaired in a coordinated manner so that the formation of DSBs is avoided. We propose that, when either base lesion is initially excised from a bistranded base damage site, the remaining base lesion will only rarely be converted into an AP site or a single strand break in vivo.

Topics: DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase; DNA, Bacterial; DNA, Single-Stranded; Escherichia coli; Escherichia coli Proteins; Guanine; Mutation; Thymine

The potential for genetic change arising from specific single types of DNA lesion has been thoroughly explored, but much less is known about the mutagenic effects of DNA lesions present in clustered damage sites. Localized clustering of damage is a hallmark of certain DNA-damaging agents, particularly ionizing radiation. We have investigated the potential of a non-mutagenic DNA base lesion, 5,6-dihydrothymine (DHT), to influence the mutagenicity of 8-oxo-7,8-dihydroguanine (8-oxoG) when the two lesions are closely opposed. Using a bacterial plasmid-based assay we present the first report of a significantly higher mutation frequency for the clustered DHT and 8-oxoG lesions than for single 8-oxoG in wild-type and in glycosylase-deficient strains. We propose that endonuclease III has an important role in the initial stages of processing DHT/8-oxoG clusters, removing DHT to give an intermediate with an abasic site or single-strand break opposing 8-oxoG. We suggest that this mutagenic intermediate is common to several different combinations of base lesions forming clustered DNA damage sites. The MutY glycosylase, acting post-replication, is most important for reducing mutation formation. Recovered plasmids commonly gave rise to both wild-type and mutant progeny, suggesting that there is differential replication of the two DNA strands carrying specific forms of base damage.

Topics: Deoxyribonuclease (Pyrimidine Dimer); DNA Damage; DNA Glycosylases; DNA-Formamidopyrimidine Glycosylase; Escherichia coli; Escherichia coli Proteins; Guanine; Mutagenesis; Mutation; Thymine

The induction of base damage products in gamma-irradiated DNA, hydrated between 2.5 and 32.8 moles of water per mole of nucleotide (tau), was investigated using the gas chromatography/mass spectrometry-selected ion monitoring technique. In general, the yields of the measured base damage products were found to be dependent on the extent of the hydration when the DNA was irradiated under nitrogen. At low hydrations (tau < or = 13), the highest yields of the measured products were found for 7,8-dihydro-8-oxo-guanine, 5,6-dihydrothymine and, to a lesser extent, 2,6-diamino-4-oxo-5-formamidopyrimidine, products which are consistent with the base radicals found in low-temperature ESR studies. At higher hydrations (tau < or = 13), changes in DNA conformation and an increase in the attack of bulk water radicals on DNA play a significant role in the formation of radiation-induced DNA base damage products. Additional findings in our study include: (1) the sum of the yields of the products formed from electron-loss centers is greater than the sum of the yields of the products formed from electron-gain centers, indicating that there might be other electron-gain products which have not been identified; (2) the combined yield for the base damage products and the release of unaltered bases at tau < or = 13 is constant, implying that radiation damage in the tightly bound water molecules of the primary hydration layer causes DNA damage (quasi-direct effect) that is similar to the damage caused by direct ionization of the DNA (direct effect); and (3) the yields of the individual base damage products that were formed from electron-loss centers can be modeled on the basis of both the known reactions that lead to the formation of the initial charged base radicals in irradiated DNA, and the known reactions that involve the conversion of these initial DNA radicals into their respective nonradical end products.

Topics: Adenine; Animals; Base Composition; Cytosine; DNA; DNA Damage; Electron Spin Resonance Spectroscopy; Electrons; Gas Chromatography-Mass Spectrometry; Guanine; Hydrolysis; Nucleic Acid Conformation; Pyrimidines; Salmon; Thymine