Academic journal article Genetics

Double-Strand Break Repair Pathways Protect against CAG/CTG Repeat Expansions, Contractions and Repeat-Mediated Chromosomal Fragility in Saccharomyces Cerevisiae

Academic journal article Genetics

Double-Strand Break Repair Pathways Protect against CAG/CTG Repeat Expansions, Contractions and Repeat-Mediated Chromosomal Fragility in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

Trinucleotide repeats can form secondary structures, whose inappropriate repair or replication can lead to repeat expansions. There are multiple loci within the human genome where expansion of trinucleotide repeats leads to disease. Although it is known that expanded repeats accumulate double-strand breaks (DSBs), it is not known which DSB repair pathways act on such lesions and whether inaccurate DSB repair pathways contribute to repeat expansions. Using Saccharomyces cerevisiae, we found that CAG/CTG tracts of 70 or 155 repeats exhibited significantly elevated levels of breakage and expansions in strains lacking MRE11, implicating the Mre11/Rad50/Xrs2 complex in repairing lesions at structure-forming repeats. About two-thirds of the expansions that occurred in the absence of MRE11 were dependent on RAD52, implicating aberrant homologous recombination as a mechanism for generating expansions. Expansions were also elevated in a sae2 deletion background and these were not dependent on RAD52, supporting an additional role for Mre11 in facilitating Sae2-dependent hairpin processing at the repeat. Mre11 nuclease activity and Tel1-dependent checkpoint functions were largely dispensable for repeat maintenance. In addition, we found that intact homologous recombination and nonhomologous end-joining pathways of DSB repair are needed to prevent repeat fragility and that both pathways also protect against repeat instability. We conclude that failure of principal DSB repair pathways to repair breaks that occur within the repeats can result in the accumulation of atypical intermediates, whose aberrant resolution will then lead to CAG expansions, contractions, and repeat-mediated chromosomal fragility.

CAG/CTG trinucleotide repeat (TNR) expansions form the basis of 14 inherited genetic disorders including Huntington's disease (HD), myotonic dystrophy type 1 (DM1), and several types of spinocerebellar ataxias (Pearson et al. 2005; Orr and Zoghbi 2007). Normal individuals carry an allele length of 6-35 repeats, which are stable. When the repeats expand beyond a threshold length of 36-40 repeats, they are unstable and become prone to subsequent expansions, and sizes of up to 135 and ~2000 CAG/CTG repeats have been documented in individuals with HD and DM1 disorders, respectively (Pearson et al. 2005). Increases in repeat length during intergenerational transmission causes increased disease severity or earlier age of onset in the offspring. In addition, expansions during the lifespan of an individual may hasten disease progression. Thus, it is of interest to elucidate the underlying mechanisms that cause CAG/CTG repeat expansions to better understand the phenomena of disease inheritance, onset, and progression. In addition, because TNRs can inhibit DNA replication and repair, they serve as a good model to study DNA repair pathways that operate at structure-forming sequences.

Due to the intrinsic nature of single-stranded CAG and CTG repeat sequences to spontaneously form stable DNA secondary structures (McMurray 1999), they can interfere withDNAreplication (LenzmeierandFreudenreich 2003), and studies in model organisms have implicated DNAreplication inCAG/CTGrepeat expansions and contractions (Lenzmeier and Freudenreich 2003; Pearson et al. 2005; Mirkin 2007). When trinucleotide repeats are replicated such that the CTG strand is on the lagging strand template, deletions are common, suggesting that hairpins form on template DNA that is transiently single-stranded during passage of the replication fork (Freudenreich et al. 1997). CTG hairpins that form on the 59 flap of an Okazaki fragment during replication or during gap repair are poor substrates for the FEN1 59 flap-cleaving endonuclease, which can lead to ligation of the unprocessed flaps and repeat expansion (Spiro et al. 1999; Henricksen et al. 2000; Liu et al. 2004; Refsland and Livingston 2005). Slippage at the 39 end of a strand during DNA replication or postreplication repair is another mechanism that can promote repeat expansions (Daee et al. …

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