Stoichiometry of Base Excision Repair Proteins Correlates with Increased Somatic CAG Instability in Striatum over Cerebellum in Huntington's Disease Transgenic Mice

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Abstract
Huntington's disease (HD) is a progressive neurodegenerative disorder caused by expansion of an unstable CAG repeat in the coding sequence of the Huntingtin (HTT) gene. Instability affects both germline and somatic cells. Somatic instability increases with age and is tissue-specific. In particular, the CAG repeat sequence in the striatum, the brain region that preferentially degenerates in HD, is highly unstable, whereas it is rather stable in the disease-spared cerebellum. The mechanisms underlying the age-dependence and tissue-specificity of somatic CAG instability remain obscure. Recent studies have suggested that DNA oxidation and OGG1, a glycosylase involved in the repair of 8-oxoguanine lesions, contribute to this process. We show that in HD mice oxidative DNA damage abnormally accumulates at CAG repeats in a length-dependent, but age- and tissue-independent manner, indicating that oxidative DNA damage alone is not sufficient to trigger somatic instability. Protein levels and activities of major base excision repair (BER) enzymes were compared between striatum and cerebellum of HD mice. Strikingly, 5′-flap endonuclease activity was much lower in the striatum than in the cerebellum of HD mice. Accordingly, Flap Endonuclease-1 (FEN1), the main enzyme responsible for 5′-flap endonuclease activity, and the BER cofactor HMGB1, both of which participate in long-patch BER (LP–BER), were also significantly lower in the striatum compared to the cerebellum. Finally, chromatin immunoprecipitation experiments revealed that POLβ was specifically enriched at CAG expansions in the striatum, but not in the cerebellum of HD mice. These in vivo data fit a model in which POLβ strand displacement activity during LP–BER promotes the formation of stable 5′-flap structures at CAG repeats representing pre-expanded intermediate structures, which are not efficiently removed when FEN1 activity is constitutively low. We propose that the stoichiometry of BER enzymes is one critical factor underlying the tissue selectivity of somatic CAG expansion. Huntington's disease (HD) is a neurodegenerative disorder that belongs to a family of genetic diseases caused by abnormal expansion of CAG/CTG repetitive sequences. The instability of trinucleotide repeat expansions in germline and somatic cells has deleterious clinical consequences in HD. For instance, transmission of longer repeats to offspring results in an earlier onset of disease, where extensive somatic expansion in the striatum, the brain region primarily affected in HD, is proposed to accelerate disease pathology. Thus, understanding the mechanisms of trinucleotide repeat instability is a major interest. We have examined the role of oxidative DNA damage and base excision repair (BER) in somatic instability, which is tissue-selective and age-dependent. We show that oxidative DNA lesions abnormally accumulate at CAG expansions in a length-dependent, yet age- and tissue-independent manners, likely due to the secondary structures formed by CAG repeats that limit access of enzymes initiating BER. In addition, our data indicate that repair by BER enzymes of some of the accessible lesions results in somatic expansion when the ratio of FEN1 to POLβ is low, as found to occur in the striatum. Our results support BER enzyme stoichiometry as a contributor to the tissue selectivity of somatic CAG expansion in HD.