We have developed a convenient experimental system to analyze large-scale repeat expansions in yeast. Differently from previously described assays, it allows one to monitor expansions of the premutation-size (78-to-150 copies) repeats well into the disease range (200-to-450 copies), providing a unique opportunity to study characteristics and genetic controls of large-scale expansions. This study investigates the mechanisms and consequences of expansions of (GAA)n
repeats, which are responsible for Friedreich’s ataxia, the most common hereditary ataxia in humans (Pandolfo, 2002
). In the other experimental yeast system that monitors expansions of up to 25 copies of triplet repeats (Miret et al., 1998
; Pelletier et al., 2003
), expansions of (GAA)n
repeats were never detected (Robert Lahue, personal communication). This might indicate that the GAA repeats should reach a higher threshold length to undergo further expansions. In our system, we have found that GAA repeats longer than 78 copies can expand.
The propensity to expand increases exponentially with an increase in repeat length (). Specifically, doubling the size of the (GAA)n repeat from 78 to 150 copies led to a three orders of magnitude increase in its expansion rate. This dramatic difference can be explained by: (i) a length-dependent increase in the propensity of a repeat to expand, or (ii) a decrease in the number of expansion steps required for longer repeats to reach the selection threshold, or (iii) the combination of both factors. Analysis of expansions of 150 GAA repeats hinted to the existence of an incremental step of the expansions, corresponding to roughly 1.5-times the repeat size. If it is applicable to repeats of other lengths, short GAA repeats would need two or more steps to reach the selection cutoff. We suspect that this is the likely explanation of their much lower expansion rates compared to the long repeats in our system. Experiments are under way to obtain unbiased length distributions for other expanded repeats by making subtle adjustments to the overall length of introns in our cassettes with various expandable repeats.
One of the unexpected observations in our study was the lack of orientation dependence for the GAA repeat expansions. Similar to what was reported previously (Krasilnikova and Mirkin, 2004
), the replication fork stalled at the repeat in one orientation only, when the GAA run was a part of the lagging strand template (). However, the rates of large-scale expansion in both orientations were quite similar (). These results are different from our previous data that expansions of long GAA repeats depended on their orientation within a plasmid (Krasilnikova and Mirkin, 2004
). This discrepancy could be due to the fact that our earlier observations were made primarily for small expansions of a much longer GAA repeat (280 copies) positioned within a multi-copy plasmid, while our current system monitors large expansions of shorter, GAA repeats on a yeast chromosome.
The first-round genetic screening gave us important clues to the mechanisms of repeat expansions. There were no differences in the expansion rates between the wild type and Rad52 knockout strains, while the rate of expansions in hyper-recombinagenic Δsgs1
mutant (Watt et al., 1996
) decreased. Therefore, the involvement of genetic recombination in the large-scale expansions of GAA repeats can be ruled out, contrary to what was described in a bacterial system (Napierala et al., 2004
). We also did not see much effect of the Msh2 protein on the GAA repeat expansions both in this and previous (Kim et al., 2008
) studies, making the mismatch repair system an unlikely player in the repeat expansion process.
Disruption of the TOF1
genes led to a strong elevation in the repeat expansion rates. These genes encode components of the so-called replication-pausing complex (Tof1-Mrc1-Csm3) (Katou et al., 2003
) that prevents the replication forks stalling caused by hydroxyurea treatment (Calzada et al., 2005
; Nedelcheva et al., 2005
), DNA damage (Foss, 2001
), or unusual DNA structures (Voineagu et al., 2008
) by averting uncoupling of the replicative DNA helicases from stalled forks (Nedelcheva et al., 2005
). In addition, Tof1p facilitates the replication fork pausing at protein-mediated barriers (Mohanty et al., 2006
In contrast, disruption of the SGS1
, or RAD5
genes inhibited repeat expansions. The SGS1
gene encodes a 3’-to-5’ DNA helicase homologous to the human Bloom’s syndrome DNA helicase (Gangloff et al., 1994
mutants are hypersensitive to UV light and hydroxyurea (Chakraverty et al., 2001
), and display hyper-recombination phenotype (Watt et al., 1996
). The Sgs1 protein was implicated in the restart of stalled replication forks (Torres et al., 2004
), and in the repair of defects accumulated during the lagging strand synthesis (Ii and Brill, 2005
). Rad6 is an ubiquitin-conjugating enzyme that acts in a complex with Rad18 ubiquitin ligase to regulate DNA damage tolerance pathway in yeast (reviewed in (Lawrence, 1994
). The latter includes a Rad5-dependent template-switching branch and the translesion DNA synthesis branch (Andersen et al., 2008
). Rad5, a member of the SWI/SNF family, has ATPase and E3 ubiquitin ligase activities (Klein, 2007
). Its ATPase activity is stimulated by the presence of branched DNA structures, which triggers a helicase-like reaction of template switching and/or fork regression (Blastyak et al., 2007
How do our results compare with the other studies on repeat expansions? In the best- characterized yeast system that dealt with smaller scale expansions of CAG repeats, Tof1 inactivation led to a 7-fold increase in the expansion rate (Razidlo and Lahue, 2008
), which is quantitatively similar to our observations. We conclude therefore, that the activity of Tof1p universally opposes small- and large-scale expansions of various triplet repeats. At the same time, our data with Sgs1, Srs2 and Rad6 knockouts contrast previous observations: disruption of the Sgs1 helicase did not affect CAG expansions (Bhattacharyya and Lahue, 2004
), while inactivation of the Srs2 DNA helicase (Bhattacharyya and Lahue, 2004
) or Rad6 repair pathway (Daee et al., 2007
) resulted in a dramatic increase in CAG expansions. These profound differences could either reflect separate mechanisms for the large- and small-scale expansions, or could be due to different structural features of CAG and GAA repeats. Future studies of CAG repeat expansions in our system could distinguish between these scenarios.
Altogether, our data left us with the following paradox. On one hand, all genes affecting GAA expansions in our system, including Tof1, Csm3, Rad6, Rad5 and Sgs1, are implicated in fork stabilization, reversal or restart. On the other hand, we do not see the link between the repeat-mediated replication fork stalling and their propensity to expand. We hypothesize that the model, loosely based on the template-switching mechanism proposed in (Goldfless et al., 2006
), could resolve this paradox. We propose that during replication of a repetitive DNA run (), a leading strand DNA polymerase can accidentally (~10−3
per replication) switch its template to continue DNA synthesis along the nascent lagging strand (). Notably in a long repetitive run, each sequence in the nascent lagging strand sequence is repeated multiple times in the leading strand template. This could make the template switch more feasible, compared to the unique DNA sequences, as an unwound portion of the repetitive leading strand can pair with multiple points along the repetitive lagging strand. After reaching the end of the Okazaki fragment (), the polymerase should switch back to its primary leading strand template in order for replication to continue. This results in an expanded repetitive run within the leading DNA strand (). These reactions would likely depend on the activities of the 3’-to-5’ DNA helicases, such as Sgs1, and the template-switcher Rad5. The Tof1/Csm3/Mrc1 fork-stabilizing complex, in contrast, would be expected to block template switching. Our data are in an agreement with these predictions.
Proposed mechanisms of expansions of the GAA repeats (see text for details)
In general, this model assumes that the maximum size of the one-step repeat expansion should be less than or equal to an Okazaki fragment. The biochemical measurement of the sizes of Okazaki fragments in eukaryotes showed that they vary significantly: from 40 to 290 nt (Anderson and DePamphilis, 1979
; Raschle et al., 2008
). Our average expansions (50-to-70 triplet repeats) are within these ranges. How can we explain bigger-size expansions also observed in our experiments? We believe that an expansion cycle presented in our model could recur more than once, particularly when the repeat’s size exceeds that of an Okazaki fragment. This could account to the largest-scale expansions observed in our hands as well as for the so-called catastrophic expansions observed for GAA repeats in human pedigrees (Montermini et al., 1997
Importantly, this rare sequence of events should not be linked to the much more frequent fork stalling at the repeat. The latter is due to the formation of the triplex DNA structure, when the homopurine run is situated on the lagging strand template during DNA replication (Krasilnikova and Mirkin, 2004
). We have previously found that such stalling results in repeat contractions, chromosomal fragility and chromosomal rearrangements mediated by the mismatch repair machinery (Kim et al., 2008
). In the current study, we observed that the formation of interstitial deletions, but not expansions, depended on the presence of functional Msh2 and Rad52 proteins. Overall, we believe that interstitial deletions result from the double-stranded breaks at GAA repeats mediated by the MMR proteins followed by their repair via single-strand annealing (SSA).
We are fully aware that the proposed model for repeat expansions is by no means final and future studies including full-genome screening are needed to gain a better insight in the mechanisms of this process. The immediate conceptual advance of our model is that it is applicable for a variety of DNA repeats, notwithstanding of their specific secondary structures. This could nicely explain how similar expansion principles apply to such structurally different repeats, including quadruplex-forming CGG repeats, hairpin-forming CTG repeats, triplex-forming GAA repeats or DNA-unwinding ATTCT repeats. At the same time, not every repetitive sequence is known to expand (McMurray, 1999
). This could be explained if a propensity for the template switching also depends on certain biophysical properties of repetitive DNAs, such as the “stickiness” characteristic of long (GAA)n
repeats (Sakamoto et al., 2001