OGG1 initiates age-dependent CAG trinucleotide expansion in somatic cells

Abstract

Although oxidative damage has long been associated with ageing and neurological disease, mechanistic connections of oxidation to these phenotypes have remained elusive. Here we show that the age-dependent somatic mutation associated with Huntington's disease occurs in the process of removing oxidized base lesions, and is remarkably dependent on a single base excision repair enzyme, 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1). Both in vivo and in vitro results support a 'toxic oxidation' model in which OGG1 initiates an escalating oxidation-excision cycle that leads to progressive age-dependent expansion. Age-dependent CAG expansion provides a direct molecular link between oxidative damage and toxicity in post-mitotic neurons through a DNA damage response, and error-prone repair of single-strand breaks.

Figure 1. Oxidative lesions accumulate in tissues of ageing mice

a, Age-dependent CAG repeat distribution in the tissues of R6/1 transgenic mice at indicated ages. The vertical line designates the midpoint length of the CAG repeat distribution in the tail of tested animals. Expansion is an increase in the number of CAG repeats indicated by the shift of distributions to the right (x axis is length in base pairs). b, Left panel, level of oxidative lesions in the tail (t), brain (br) (cortex) and liver (lv) for 8-oxo-G in control (Ctrl) and R6/1 animals at 7 (black) and 52 (grey) weeks. Right panel, accumulation (fold change) of the number of lesions from 7 to 52 weeks. Error bars, s.d. c, Accumulation as in b for 5-OH-uracil, 3-meA and uracil. ★P < 0.01 for b and c. d, Repair activity (Methods) of 8-oxo-G DNA lesion in R6/1 animals (black circles) and wild-type littermate control (open circles) does not change with age (weeks) e, Quantified repair activity (%) of 8-oxo-G, 5-OHC, 3-meA, FAPY and uracil as in d for the indicated tissues at 7 (black) and 52 (grey) weeks. Reported are the mean repair activity (%) and the s.d. The limit of the s.d. is 50 (3-meA).

Figure 2. Direct exposure to oxidizing agents causes expansion at the human HD locus in vitro.

aa, CAG repeat distribution of pure sorted cortical neurons at 27 weeks as compared with that of the tail at 3 weeks and to whole-cortex cell suspensions of the same animal before sorting. Vertical dashed line designates the midpoint length of the CAG repeat tract in the tail. Numbers represent size standards. b, The quantified sizes of the repeat tract in tail at 3 weeks (light grey), cortex mixture (dark grey) and pure sorted neurons at 27 weeks (black). Error bars, s.d.; Δ, mean length change of CAG repeats. c–e, Fibroblasts (c and e) and lymphoblasts (d) from Huntington’s disease patients treated in culture with indicated concentrations of hydrogen peroxide. The expanded allele is 69 repeats and the normal allele is 16 CAG repeats. Expansion in peroxide-treated cells versus untreated cells (Ctrl) was determined as in Fig. 1. f, Comet assay for SSBs. Increasing peroxide treatment as indicated induces SSB as detected by comet tails. SSBs were repaired in cells by 2 h post treatment (5 mM H₂O₂/recovery) as judged by the loss of the comet tails.

Figure 3. Age-dependent expansion is suppressed in mice lacking OGG1 glycosylase

a, b, Representative CAG repeat distributions in the tissues of R6/1 transgenic or R6/1/OGG1^−/− mice. a, Vertical dashed line designates the midpoint of the CAG repeat tract length in the tail at 3 weeks (t1, white box). The CAG tract lengths in the tail (t2, light grey), brain (br, dark grey) (cortex) and liver (lv, black) at 25 weeks in the same animal are shown. b, The mean length change of CAG repeats (Δ) at the indicated ages in the tissues of R6/1 mice and R6/1/OGG1^−/− mice. Each value is expressed as the mean change and the s.d. Tissues are indicated by colour: tail at 3 weeks in white; brain and liver at indicated ages in dark grey and in black respectively. c, Quantified data for mean length change of CAG repeats from R6/1 mice, R6/1/AAG^−/− or R6/1/NTH^−/−. Tissues are indicated by colour: tail at 3 weeks in white; tail, brain and liver at 27 weeks in light grey, dark grey and black respectively. Analysis is the same as in a, b.

Figure 4. OGG1 excision of 8-oxo-G within CAG repeat DNA can initiate strand displacement and expansion in vitro during BER

a, Reaction products after step-wise addition of OGG1 (lanes 1 and 4), OGG1 + APE (lanes 2 and 5), and OGG1 + APE + Polβ (lanes 3 and 6) to random (left) or CAG (right) templates. Red dots indicate 22 nucleotide excision/incision product, open and filled circles are 1 nucleotide and 3 nucleotide additions, respectively. b, Triplet pattern on CAG template as a function of increasing Polβ concentration indicated by the triangle: 0 nM (lane 7); 0.5 nM (lane 8); and 1 nM (lane 9). c, Products of the BER reactions for random (left) or CAG (right) templates in the absence (−) or presence (+) of DNA ligase. FL, 100 nucleotide full-length product after ligation. The bracket and asterisk depict the 100 nucleotide FL template and larger expansion products. Red dot as in a. d, ‘Toxic oxidation cycle’ model for age-dependent somatic expansion. Endogenous oxidative radicals (O°) arising from mitochondrial (MT) respiration creates oxidative DNA lesions. Under conditions of normal BER, OGG1/APE cleavage produces a nick, and polymerase (Pol) facilitates hairpin formation during gap-filling synthesis. CAG hairpins are stabilized by MSH2/MSH3 binding (red dot is a mismatch in the stem) and escape FEN-1 loading and cleavage owing to a hidden 5′ end. The hairpin intermediate is processed to restore duplex DNA generating a longer CAG template, which is again subject to oxidative DNA damage. The cycle continues with age.