Abstract
Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases.
Highlights
Repetitive DNA is found dispersed throughout eukaryotic genomes, and in some cases is central to key biological processes such as chromosome segregation and chromosome end protection [1]
Expansion of a CAG/CTG trinucleotide repeat is the causative mutation for multiple neurodegenerative diseases, including Huntington’s disease, myotonic dystrophy, and multiple types of spinocerebellar ataxias
We show that an expanded CAG/CTG tract placed within the chromosome of the model eukaryote, budding yeast, elicits a cellular response that interferes with cell growth and division
Summary
Repetitive DNA is found dispersed throughout eukaryotic genomes, and in some cases is central to key biological processes such as chromosome segregation and chromosome end protection [1]. Expansion of CAG/CTG trinucleotide repeats (abbreviated CAG) have been observed to occur at several different genomic loci, causing diseases that include Huntington’s disease, myotonic dystrophy, and multiple subtypes of spinal cerebellar ataxia [2,3]. In addition to the instability threshold, a disease-causing threshold exists for trinucleotide repeats, which is at or above the expansion threshold, and is dependent on the locus and disease process. For Huntington’s disease the disease-causing threshold is 38–40 repeats, and is governed by the length at which the polyglutamine tract (coded for by CAG) within the Huntingtin gene becomes toxic. At the myotonic dystrophy locus, the disease threshold is closer to 200 repeats, the size at which the CUG RNA exerts toxic effects on muscle cells [2,4]
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