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. 2011 Jul 8;410(2):183-93.
doi: 10.1016/j.jmb.2011.05.004. Epub 2011 May 13.

Escherichia coli Fpg glycosylase is nonrendundant and required for the rapid global repair of oxidized purine and pyrimidine damage in vivo

Brandy J Schalow  1 , Charmain T Courcelle, Justin Courcelle
Affiliations

Escherichia coli Fpg glycosylase is nonrendundant and required for the rapid global repair of oxidized purine and pyrimidine damage in vivo

Brandy J Schalow et al. J Mol Biol. .

Abstract

Endonuclease (Endo) III and formamidopyrimidine-N-glycosylase (Fpg) are two of the predominant DNA glycosylases in Escherichia coli that remove oxidative base damage. In cell extracts and purified form, Endo III is generally more active toward oxidized pyrimidines, while Fpg is more active towards oxidized purines. However, the substrate specificities of these enzymes partially overlap in vitro. Less is known about the relative contribution of these enzymes in restoring the genomic template following oxidative damage. In this study, we examined how efficiently Endo III and Fpg repair their oxidative substrates in vivo following treatment with hydrogen peroxide. We found that Fpg was nonredundant and required to rapidly remove its substrate lesions on the chromosome. In addition, Fpg also repaired a significant portion of the lesions recognized by Endo III, suggesting that it plays a prominent role in the global repair of both purine damage and pyrimidine damage in vivo. By comparison, Endo III did not affect the repair rate of Fpg substrates and was only responsible for repairing a subset of its own substrate lesions in vivo. The absence of Endo VIII or nucleotide excision repair did not significantly affect the global repair of either Fpg or Endo III substrates in vivo. Surprisingly, replication recovered after oxidative DNA damage in all mutants examined, even when lesions persisted in the DNA, suggesting the presence of an efficient mechanism to process or overcome oxidative damage encountered during replication.

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Figures

Figure 1
Figure 1. The structure and relative abundance of some common forms of oxidized base adducts in H2O2-treated cultures
aApproximate frequency of lesions based on treatment with increasing concentrations of hydrogen peroxide.
Figure 2
Figure 2. In vitro, the substrate specificity of Fpg and Endo III partially overlap. However, Fpg is more active at 8-oxoguanines and Endo III is more active at thymine glycols
Purified high molecular weight DNA containing no lesions, oxidized purines (methylene blue + light treated), or oxidized pyridines (osmium tetroxide treated) was incubated for 1 hr in the presence of no glycosylase, Fpg glycosylase, or Endo III as indicated and analyzed by alkali-agarose gel electrophoresis. The presence of DNA lesions that are recognized by either Fpg or Endo III is indicated by the loss of high molecular weight DNA fragments in those samples. Lane M - lambda HindIII size marker; Fpg - Fpg glycosylase; EndoIII - Endo III glycosylase.
Figure 3
Figure 3. Fpg is required to rapidly remove oxidized purines and pyrimidines from the global genome in vivo
A) Lesions recognized by Fpg and Endo III are rapidly repaired from wild-type cultures in vivo. Cultures were exposed to 10 mM hydrogen peroxide for 5 min and allowed to recover. At the times indicated, genomic DNA was purified, incubated for 1hr in the presence of no glycosylase, Fpg, or Endo III, then analyzed by alkali-agarose gel electrophoresis. The loss of high molecular weight DNA fragments following glycosylase treatment indicates the presence of Fpg- or Endo III-recognized lesions in the DNA. Lane M - lambda HindIII size marker. B) Fpg is required to rapidly remove both Fpg- and Endo III-recognized lesions from the E. coli genome. Cultures of fpg and nth mutants were treated and analyzed as in (A). C) The percent lesion-free, high molecular weight DNA in Fpg- (open circles) and Endo III-treated (filled triangle) samples is plotted at each time point relative to mock-treated samples. All plots represent an average of three independent experiments. Error bars represent the standard error of the mean.
Figure 4
Figure 4. Endo VIII and nucleotide excision repair do not significantly contribute to the removal of 8-oxoguanine or thymine glycol lesions from the overall genome
A) nei (upper panel) and uvrA (lower panel) cultures were exposed to 10 mM hydrogen peroxide for 5 min and allowed to recover. At the times indicated, genomic DNA was purified, incubated for 1 hr in the presence of no glycosylase, Fpg, or Endo III, then analyzed by alkali-agarose gel electrophoresis. Lane M - lambda HindIII size marker. B) The percent of lesion-free, high molecular weight DNA in Fpg- (open circles) and Endo III-treated (filled triangle) samples is plotted at each time point relative to mock-treated samples. All plots represent an average of three independent experiments. Error bars represent the standard error of the mean.
Figure 5
Figure 5. The absence of Fpg, Endo III, Endo VIII or nucleotide excision repair does not prevent DNA synthesis from resuming following hydrogen peroxide-induced DNA damage
A) Cultures grown in the presence of 3H-thymine were exposed to hydrogen peroxide for 5 min (open circles) or mock-treated (filled triangles), resuspended in fresh 37°C medium and allowed to recover. The relative amount of 3H-thymine incorporated into the DNA is plotted over time. All plots represent an average of two independent experiments. Error bars represent the standard error of the mean. The amount of 3H in each sample at time 0 was between 7000 and 13000 cpm for all experiments. B) Cells lacking DNA glycosylases or nucleotide excision repair are not hyper-sensitive to hydrogen peroxide. The survival of each strain following exposure to 10 mM hydrogen peroxide for the indicated time period is plotted. Strains: wild-type (open square), Δfpg (CL1009, filled circle), Δnei (CL 1005, filled diamond), Δnth (CL1006, filled triangle), ΔuvrA (CL21, filled square), and ΔrecA (CL002, open circle). Plots represent an average of three independent experiments. Error bars represent the standard error of the mean.
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