Deletion of OGG1 Results in a Differential Signature of Oxidized Purine Base Damage in mtDNA Regions

Abstract

Mitochondrial oxidative stress accumulates with aging and age-related diseases and induces alterations in mitochondrial DNA (mtDNA) content. Since mtDNA qualitative alterations are also associated with aging, repair of mtDNA damage is of great importance. The most relevant form of DNA repair in this context is base excision repair (BER), which removes oxidized bases such as 8-oxoguanine (8-oxoG) and thymine glycol through the action of the mitochondrial isoform of the specific 8-oxoG DNA glycosylase/apurinic or apyrimidinic (AP) lyase (OGG1) or the endonuclease III homolog (NTH1). Mouse strains lacking OGG1 (OGG1^-/-) or NTH1 (NTH1^-/-) were analyzed for mtDNA alterations. Interestingly, both knockout strains presented a significant increase in mtDNA content, suggestive of a compensatory mtDNA replication. The mtDNA "common deletion" was not detected in either knockout mouse strain, likely because of the young age of the mice. Formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites accumulated in mtDNA from OGG1^-/- but not from NTH1^-/- mice. Interestingly, the D-loop region was most severely affected by the absence of OGG1, suggesting that this region may be a hotspot for oxidative damage. Thus, we speculate that mtDNA alterations may send a stress message to evoke cell changes through a retrograde mitochondrial-nucleus communication.

Keywords: 8-oxoG localization; NTH1 ko; OGG1 knockout (ko); mtDNA content; mtDNA repair.

Figure 1

Relative mitochondrial DNA (mtDNA) content in liver from wild-type (wt), endonuclease III homolog knockout (NTH1^−/−), and mitochondrial isoform of 8-oxoG DNA glycosylase/apurinic or apyrimidinic (AP) lyase knockout (OGG1^−/−) mice. Bars represent the mean value and standard error of the mean (SEM) of the relative mtDNA content determined by qPCR of two independent experiments conducted in triplicates. Statistical difference was determined by the Kruskal–Wallis test with the Dunn’s multiple comparison test. Bars not sharing a common superscript letter differ significantly (p < 0.05, Dunn’s multiple comparison test); n = number of analyzed animals.

Figure 2

End-point PCR analysis of mouse mtDNA “common deletion”. Representative gel of end-point PCR amplification of total DNA isolated from liver of three 18-month-old wt mice, and three 4-month-old mice, belonging to wt, NTH1^−/−, and OGG1^−/− strains, respectively. (A) extension step = 4 min; (B) extension step = 30 s. Amplicons (5-μL aliquots) were visualized by gel electrophoresis on ethidium bromide-stained 1.5% agarose gel. Lane loading was as follows: a = EZ load 500-bp molecular ruler (BioRad Laboratories Inc., Hercules, CA, USA); b, c, d = total DNA from each one of the three 18-month-old wt mice; e, f, g, = total DNA from one wt, one NTH1^−/−, and one OGG1^−/− four-month-old mouse, respectively; h = blank with no DNA template; i, GeneRuler 100-bp DNA ladder (Thermo Fisher Scientific Inc, Waltham, MA, USA); j, k, l = total DNA from each one of three 18-month-old wt mice; m, n, o = total DNA from one wt, one NTH1^−/−, and one OGG1^−/− four-month-old mouse, respectively; p = blank with no DNA template.

Figure 3

Analysis of purine-specific mtDNA damage at the d-loop, Ori-l, ND1, and direct repeat 1 (DR1) regions in liver from wt, NTH1^−/−, and OGG1^−/− mice. (A) Map of mouse mtDNA. The thick arches extending out of the circle represent the four amplified regions tested for formamidopyrimidine DNA glycosylase (Fpg) sensitivity delimited, respectively, by the primer pairs listed in Table 2, and corresponding to the indicated nucleotides. Numbering is according to National Center for Biotechnology Information (NCBI) accession number NC_005089.1. The outmost arch represents the 3.8-kb deletion, delimited by direct repeat 1 (DR1) and direct repeat 2 (DR2) represented by two full squares. (B) representative gel analysis of amplicons from Fpg-treated and untreated total DNA from one wt and one OGG1^−/− mouse (the target was the d-loop region). An aliquot (5 μL) of each PCR reaction was loaded onto a 1.5% agarose ethidium bromide-stained gel and analyzed for band intensities. Lane loading was as follows: a = GeneRuler 100-bp DNA ladder (Thermo Fisher Scientific Inc, Waltham, MA, USA); b, c, e, f, h, i, k, and l = 5 ng of total DNA as template; d, g, j, and m = 7.5 ng of total DNA as template. (C) Bars represent the mean and SEM of the ratio between Fpg-treated and untreated band intensities, expressed as the percentage of the complement to 100 to improve the graphical evaluation. Statistical difference determined by the Kruskal–Wallis test with Dunn’s multiple comparison test. Bars not sharing a common superscript letter differ significantly (p < 0.05).