Abstract
Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER). We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA)20 repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol β DNA synthesis and the formation of a short 5'-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5'-GAA repeat flap. Pol β then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA patient cells. This further suggests the potential of developing chemotherapeutic alkylating agents to shorten expanded GAA repeats for treatment of FRDA.
Highlights
Friedreich’s ataxia (FRDA, OMIM 229300) is one of the most prevalent inherited autosomal recessive neurodegenerative disorders
To determine whether alkylated DNA base lesions induced in the intronic expanded generated [56]. For a (GAA) repeat tracts can result in GAA repeat instability, we initially examined the effects of temozolomide on the instability of intronic GAA repeats in lymphoblasts from both a normal individual and a FRDA patient
We provide the first evidence that the chemotherapeutic DNA damaging agent temozolomide can predominantly induce massive contractions in expanded intronic GAA repeats in FRDA lymphoblasts (Figure 1B)
Summary
Friedreich’s ataxia (FRDA, OMIM 229300) is one of the most prevalent inherited autosomal recessive neurodegenerative disorders. Because frataxin plays a crucial role in maintenance of iron homeostasis, heme biosynthesis and assembly of iron-sulfur clusters (ISCs) into metabolic enzymes [5], cellular deficiency of the protein can lead to an insufficiency of electrotransfer through a series of proteins and enzymes of the respiratory chain. This subsequently increases electron leakage that in turn results in energy deficiency and oxidative stress leading to death of large sensory neurons in the dorsal root ganglia (DRG) and the posterior columns of the spinal cord among others [6]. The extent of GAA repeat expansion correlates with disease severity and early age of onset [7,10,11]
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