Abstract
Huntington's disease (HD), a neurodegenerative disease characterized by progressive dementia, psychiatric problems, and chorea, is known to be caused by CAG repeat expansions in the HD gene HTT. However, the mechanism of this pathology is not fully understood. The translesion DNA polymerase θ (Polθ) carries a large insertion sequence in its catalytic domain, which has been shown to allow DNA loop-outs in the primer strand. As a result of high levels of oxidative DNA damage in neural cells and Polθ's subsequent involvement in base excision repair of oxidative DNA damage, we hypothesized that Polθ contributes to CAG repeat expansion while repairing oxidative damage within HTT. Here, we performed Polθ-catalyzed in vitro DNA synthesis using various CAG•CTG repeat DNA substrates that are similar to base excision repair intermediates. We show that Polθ efficiently extends (CAG)n•(CTG)n hairpin primers, resulting in hairpin retention and repeat expansion. Polθ also triggers repeat expansions to pass the threshold for HD when the DNA template contains 35 repeats upward. Strikingly, Polθ depleted of the catalytic insertion fails to induce repeat expansions regardless of primers and templates used, indicating that the insertion sequence is responsible for Polθ's error-causing activity. In addition, the level of chromatin-bound Polθ in HD cells is significantly higher than in non-HD cells and exactly correlates with the degree of CAG repeat expansion, implying Polθ's involvement in triplet repeat instability. Therefore, we have identified Polθ as a potent factor that promotes CAG•CTG repeat expansions in HD and other neurodegenerative disorders.
Highlights
The expansion of trinucleotide repeats (TNRs) is associated with a number of neurodegenerative disorders, including Huntington’s disease (HD) and myotonic dystrophy [1, 2]
To determine whether polymerase θ (Polθ) can extend a CAG or CTG hairpin primer, we conducted in vitro DNA synthesis using a limited amount of HeLa nuclear extracts supplemented with a purified 90-kD catalytic Polθ polypeptide (Fig. S1) [32], a (CAG)5 or (CTG)5 hairpin primer that anneals to ssM13mp18(CTG)10 or ssM13mp18(CAG)10 (Fig. 1B), respectively, as described previously [23]
When purified Polθ replaced polymerase β (Polβ) or Polδ in the synthesis reaction, most yielded products were the same as those observed in the Polβ reaction (Fig. 1C, lane 5)
Summary
DNA polymerase θ promotes CAGCTG repeat expansions in Huntington’s disease via insertion sequences of its catalytic domain. Chan, Xueying Li1, Janice Ortega, Liya Gu1, and Guo-Min Li1,2,* From the 1Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas, USA; 2Department of Toxicology and Cancer Biology, University of Kentucky College of Medicine, Lexington, Kentucky, USA
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