Processing of thymine glycol in a clustered DNA damage site: mutagenic or cytotoxic

Abstract

Localized clustering of damage is a hallmark of certain DNA-damaging agents, particularly ionizing radiation. The potential for genetic change arising from the effects of clustered damage sites containing combinations of AP sites, 8-oxo-7,8-dihydroguanine (8-oxoG) or 5,6-dihydrothymine is high. To date clusters containing a DNA base lesion that is a strong block to replicative polymerases, have not been explored. Since thymine glycol (Tg) is non-mutagenic but a strong block to replicative polymerases, we have investigated whether clusters containing Tg are highly mutagenic or lead to potentially cytotoxic lesions, when closely opposed to either 8-oxoG or an AP site. Using a bacterial plasmid-based assay and repair assays using cell extracts or purified proteins, we have shown that DNA double-strand breaks (DSBs) arise when Tg is opposite to an AP site, either through attempted base excision repair or at replication. In contrast, 8-oxoG opposite to Tg in a cluster 'protects' against DSB formation but does enhance the mutation frequency at the site of 8-oxoG relative to that at a single 8-oxoG, due to the decisive role of endonucleases in the initial stages of processing Tg/8-oxoG clusters, removing Tg to give an intermediate with an abasic site or single-strand break.

Figure 1.

Mutation frequency of the Go/Tg clustered damage site transformed into wild type, mutY strain and fpg mutY strains of E. coli. The types of clusters are shown along the x-axis. Error bars display the standard errors of the mean from three experiments.

Figure 2.

The effect of Tg on the time for rejoining of an AP site (A) or a HAP1-SSB (B) when in different clustered sites following incubation with xrs5 nuclear extracts. The extent of accumulation of SSB+1 base during repair of an AP site (C) or a HAP1-SSB (D) when ddATP replaces dATP in the repair buffer. AP site control (con, filled circle: solid line) and AP/Tg at positions +1 (filled square: solid line), +5 (filled triangle: solid line), −1 (filled square: dashed line), −5 (filled triangle: dashed line). To assist the reader, lines have been drawn between the points to guide the eye only and do not represent fitted curves.

Figure 3.

Effect of Tg on the incision of an AP site and addition of the first base when at positions +1 and −1 in a cluster by the proteins HAP-1 and HAP-1 + pol β, respectively.

Figure 4.

Effect of Tg on the efficiency of incision of an AP site in the different clustered sites by (A) Fpg (1 pg to 10 ng), (B) Nth (1 pg to 10 ng), (C) Nei (0.05–1 U) and (D) exo III (0.01–0.1 U) in double-stranded oligonucleotides. The fold inhibition/activation was obtained from comparison of the concentration dependence for incision of the AP site with that for the control AP site. The error bars represent the standard deviation from three different experiments.

Figure 5.

Effect of an AP site on (A) the efficiency of excision of Tg. by Nth (1 pg to 1 ng) and (B) the dependence for formation of a DSB on the concentration of Nth determined following native PAGE. Effect of 8-oxoG on the efficiency of excision of Tg. by Nth (1 pg to 10 ng) (C) or Nei (0.05–1 U) (D). Effect of Tg on the efficiency of excision of 8-oxoG by Fpg (1 pg to 10 ng) (E). The fold inhibition was obtained from comparison with the control containing only Tg (A–D) or 8-oxoG (E). The error bars represent the standard deviation from three different experiments.