Abstract
Guanine is the most easily oxidized of the four DNA bases, and contiguous guanines (GG) in a sequence are more readily oxidized than a single guanine in a sequence. Continued oxidation of GGs results in a contiguous oxidized guanine lesion. Two contiguous 2,5-diamino-4H-imidazol-4-ones, an oxidized form of guanine that hydrolyses to 2,2,4-triamino-5(2H)-oxazolone (Oz), are detected following the oxidation of GG. In this study, we analysed translesion synthesis (TLS) across two contiguous Oz molecules (OzOz) using Klenow Fragment exo− (KF exo−) and DNA polymerases (Pols) α, β, ζ, η, ι, κ and REV1. We found that KF exo− and Pols α, β, ι and REV1 inserted one nucleotide opposite the 3′ Oz of OzOz and stalled at the subsequent extension, and that Pol κ incorporated no nucleotide. Pol η only inefficiently elongated the primer up to full-length across OzOz; the synthesis of most DNA strands stalled at the 3′ or 5′ Oz of OzOz. Surprisingly, however, Pol ζ efficiently extended the primer up to full-length across OzOz, unlike the other DNA polymerases, but catalysed error-prone nucleotide incorporation. We therefore believe that Pol ζ is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ζ.
Highlights
Mutations of genetic information are caused by various endogenous and exogenous oxidative stresses, leading to carcinogenesis, aging and other diseases
We previously investigated which nucleotides are incorporated opposite a single Oz using Klenow Fragment exoÀ (KF exoÀ), Sulfolobus solfataricus DNA polymerase (Pol) IV and the eukaryotic Pols, e, and REV1 (1921)
Stalling of DNA synthesis by KF exoÀ, calf thymus Pol a and human Pol b at the OzOz lesion We examined whether KF exoÀ, and Pols and, can elongate the primer across the OzOz lesion by synthesizing a 30-mer DNA oligonucleotide containing the OzOz lesion and using it as template
Summary
Mutations of genetic information are caused by various endogenous and exogenous oxidative stresses, leading to carcinogenesis, aging and other diseases. Guanine is the most sensitive of the four DNA bases to oxidative stress [1], and oxidized guanine lesions induce G:C-T:A and G:C-C:G transversions [2]. Oz has been detected in samples of liver DNA, and photooxidation in the presence of 5-methylcytosine significantly increased the yield of Oz at the CpG site in the p53 tumour suppressor gene [18] It appears that Oz has a more significant biological effect than Iz. We previously investigated which nucleotides are incorporated opposite a single Oz using Klenow Fragment exoÀ (KF exoÀ), Sulfolobus solfataricus DNA polymerase (Pol) IV and the eukaryotic Pols , , , , e, , , , and REV1 (1921). Since Iz is an oxidation product of both guanine and 8-oxoG, the biological influence of OzOz is important and should be analysed
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