Replication in mammalian cells recapitulates the locus-specific differences in somatic instability of genomic GAA triplet-repeats

Abstract

Friedreich ataxia is caused by an expanded (GAA·TTC)n sequence in intron 1 of the FXN gene. Small pool PCR analysis showed that pure (GAA·TTC)44+ sequences at the FXN locus are unstable in somatic cells in vivo, displaying both expansions and contractions. On searching the entire human and mouse genomes we identified three other genomic loci with pure (GAA·TTC)44+ sequences. Alleles at these loci showed mutation loads of <1% compared with 6.3–30% for FXN alleles of similar length, indicating that somatic instability in vivo is regulated by locus-specific factors. Since distance between the origin of replication and the (CTG·CAG)n sequence modulates repeat instability in mammalian cells, we tested if this could also recapitulate the locus-specific differences for genomic (GAA·TTC)n sequences. Repeat instability was evaluated following replication of a (GAA·TTC)115 sequence in transfected COS1 cells under the control of the SV40 origin of replication located at one of five different distances from the repeat. Indeed, depending on the location of the SV40 origin relative to the (GAA·TTC)n sequence, we noted either no instability, predominant expansion or both expansion and contraction. These data suggest that mammalian DNA replication is a possible mechanism underlying locus-specific differences in instability of GAA triplet-repeat sequences.

Stalled DNA Replication Forks at the Endogenous GAA Repeats Drive Repeat Expansion in Friedreich’s Ataxia Cells

“…Using a plasmid-based model system, it has been shown that the replication fork stalls at expanded GAA repeats (Chandok et al, 2012; Krasilnikova and Mirkin, 2004) and that altered replication fork progression results in repeat instability (Cleary et al, 2002; Pelletier et al, 2003; Rindler et al, 2006; Shishkin et al, 2009). It is still unknown, however, how the replication fork proceeds through the endogenous FXN locus in the native chromatin environment and whether the replication machinery stalls at the GAA repeats in FRDA cells.…”

“…In plasmid-based experiments, an increase in instability of particular expansions was detected when TTC repeats were located on the lagging strand template (3’−5’ direction at the endogenous locus) (Rindler et al, 2006). To determine an accurate direction of the replication fork, we calculated the percentage of molecules with replication forks in either the 5’−3’ or 3’−5’ direction progressing through the GAA repeats in control hESCs, control iPSCs, FRDA iPSCs and FRDA fibroblasts (Figure 3F).…”

“…In plasmid-based model systems, replication fork progression from 5’−3’ through GAA repeats led to repeat contraction and expansion. However, replication forks progressing from the 3’−5’ direction (TTC sequence as the template for lagging strand synthesis) mostly resulted in repeat expansion (Rindler et al, 2006). We postulate that in FRDA iPSCs the replication fork direction is switched due to the activation of proximal replication origins downstream of the repeats.…”

“…Besides replication fork stalling, studies in model systems have shown that GAA repeat instability could depend on the orientation of replication fork movement through the repeats (Rindler et al, 2006). Several models have been proposed where activation or deactivation of replication origins changes their position and distance relative to the repeat tract (origin switch and origin shift model), which influences the replication fork direction through the repeats.…”

Progressive GAA·TTC Repeat Expansion in Human Cell Lines

“…Replication has been shown to influence GAA·TTC repeat stability in previous model systems [24], [28]–[30],[39]. To analyze the influence of replication on GAA·TTC expansion in our cell lines, we sought to alter cell division rates during culturing and analyze the influence on GAA·TTC stability (Figure 4).…”