The site-specific integration of specific repeat length (CTG)•(CAG) tracts alongside an ectopic copy of the human c-myc replication origin has allowed analysis of the effect of repeat length, replication polarity and origin location on TNR instability. The clonal origin of each of the cell lines used in this work eliminated progenitor cell heterogeneity and differences in patient age, tissue type, genetic background, and chromosomal location that may have complicated the interpretation of previous studies. In this model we observed that (CTG)•(CAG) microsatellite instability is enhanced by prolonged cell growth, increased repeat length, origin proximity, and the inhibition of replication. Furthermore, the pattern of instability was orientation dependent. When (CTG)102 comprised the lagging template strand, the repeat showed a strong bias towards contraction in vivo, whereas both contractions and expansions were observed when (CAG)102 was present in the lagging strand template.
In bacteria and yeast (CTG)•(CAG) repeat contractions occur more often when the (CTG) sequence, which exhibits greater structure-forming potential 5,24,30
, is present in the lagging strand template 31
; this effect was observed in the current study as well. It has been argued alternatively that (CAG) hairpins in the leading strand template are responsible for this phenomenon 19
. Our ZFN digestion data indicate that (CTG) or (CAG) sequences can both form hairpins in vivo in either the leading strand or lagging strand templates. On the other hand, since both (CTG) and (CAG) template sequences can form hairpins, the effect of replication orientation on the pattern of instability suggests that leading and lagging strands do not have the same tendency to expand or contract. Two or three major deletion products were identified in (CTG)102
cell DNA after extended culture or after replication inhibition. If both template strands are prone to contraction, it would not be expected that reversal of the TNR (i.e. in (CAG)102
cells) would lead to disappearance of these contraction products. Therefore, it seems likely that both contracted products come predominantly from one of the template strands. Since the ZFN digestions indicate that either template strand can form hairpins, we presume that a step after hairpin formation is responsible for the selective contraction on just one template strand.
Slowing of leading and lagging strand synthesis with aphidicolin accelerated the (CTG)102
TNR contraction and (CAG)102
TNR expansion in a manner similar to that seen after long term culture. Emetine inhibition of lagging strand DNA synthesis in (CTG)102
cells also resulted in the appearance of a small number of prominent contraction products, as did inhibition of Okazaki fragment maturation by Fen1 knockdown. Each of these inhibitors can also cause fork stalling and reversal 32
, which may explain the resemblance of the contraction products following these alternative forms of replication inhibition 33,34
. While variation in the sizes of the contraction products may be due to unique structures of the replicative intermediates induced by each inhibitor, the overall similarity in the pattern of contraction products after aphidicolin, emetine or Fen1 siRNA treatment suggests that the physical properties of the TNR are more significant in determining the results of instability than is the mechanism of replication inhibition. That TNR orientation relative to the c-myc origin affects the observed patterns of (CTG)•(CAG) instability suggests that the (CTG) and (CAG) repeats respond differently as template vs. nascent strands or leading vs. lagging strands during replication or sister chromatid homology-based repair after fork stalling.
Yang et al. have reported that aphidicolin or emetine treatment of primary fibroblasts from a fetus with DM1 strongly enhanced expansion of the (CTG)216
allele but did not affect the normal (CTG)12
. These results are in good agreement with the marked expansion induced by aphidicolin or emetine in (CAG)102
cells and the stability of (CTG)12
tracts to these drug treatments. While the direction of replication through the DMPK locus was not mapped by Yang et al., the observation that (CTG)102
tracts expand in (CAG)102
cells treated with emetine predicts that the (CTG)216
allele studied by Yang et al. was preferentially replicated from a downstream origin, such that the (CAG) sequence was replicated as the lagging strand template.
Replication based models posit that expansion occurs because of hairpin formation in lagging strand Okazaki fragments, or in leading strand nascent DNA during fork reversal 32,35
. Several laboratories have attempted to address the role of Okazaki fragment maturation in TNR instability by inhibiting Fen1 nuclease, yet the consequences of Fen1 knockdown appear to be cell type specific 15–17,31,36–38
. Variability in the chromosomal location, repeat length, and amount of natural flanking sequence 39
may also complicate interpretation of these results. In this regard, the fact that the (CTG)•(CAG) sequences used here contained less than 20 bp of 5’ or 3’ DMPK flanking sequences implies that the present results reflect the inherent characteristics of the (CTG)•(CAG) TNR with respect to length and replication polarity separate from significant shielding effects of DMPK flanking DNA.
Arguing against a major a role for Okazaki fragment maturation in (CTG)•(CAG) expansion, the most dramatic increases in expansions occurred when Okazaki fragment synthesis was inhibited by emetine, which induced two major expanded products in (CAG)102
cells and a complex array of expansion products in (CTG)102
cells. Under these conditions an extended single strand lagging template may potentiate fork reversal and hairpin formation in the displaced leading strand nascent DNA 32,35
. The extruded nascent strand could then form a variety of stable (CTG) hairpin forms in (CTG)102
cells but a more limited array of stable (CAG) hairpins in (CAG)102
cells. The presence of even fewer contractions in (CAG)102
cells following emetine treatment compared to Fen1 siRNA or aphidicolin treatment supports the notion that the (CAG) repeats rarely contract in the lagging strand template and (CTG) repeats rarely contract in the leading strand template. The absence of contractions in (CAG)102
cells following emetine treatment may also point to a role for Okazaki fragment synthesis in stabilizing lagging strand (CAG) hairpins.
In the ectopic c-myc origin system, removal of the DUE eliminates origin activity 22
and (CTG)•(CAG) TNR instability. The reasons for the correlation between origin location and TNR instability may not be related to proximal origin activity, but may be due to a change in the replication polarity of the repeats (ori-switch hypothesis 9
), a result of the origin presenting a distinctive chromatin or DNA structure, or a more circumscribed zone for the initiation of Okazaki fragment synthesis relative to the TNR (ori-shift hypothesis 13
). Alternatively, the structure of the replication fork or the composition of the replisome may change as it moves from the origin.
HD or DM1 (CTG)•(CAG) microsatellites in humans and transgenic mice show varying degrees of expansion bias 15,17,40,41
, while analyses in bacteria, yeast, and human cultured cells reveal preferential contractions 15,42,43
. The ectopic (CTG)102
cell lines used here showed primarily contractions, although occasional expansions were observed upon long-term culture of (CAG)102
cells. Similarly, DM1 lymphoblastoid cell lines (LBCLs) demonstrate preferential contractions of the progenitor repeat length 42
. Upon continued culture, LBCLs containing the contracted alleles disappear from the population, and are replaced by more rapidly proliferating mutants with large repeat expansions 42
. These results evoked the hypothesis that mitotic drive may contribute to the expansion bias observed at the tissue level. During unperturbed cell division (CTG)102
cells most often experience TNR contractions upon extended culture, while three different means of replication inhibition rapidly induced repeat contraction in (CTG)102
cells and expansion in (CAG)102
cells. These observations, and the report that the aphidicolin and emetine regimens used here do not select for drug resistant cell lines 12
, suggest that the induced instability results from aberrant DNA synthesis rather than growth selection.
Extended passage (CTG)102
cells accumulate primarily contractions in the (CTG)•(CAG) TNR. The replication-dependent sensitivity of the (CTG)•(CAG) TNR to cleavage by either ZFNCTG
in short term passage (CTG)102
cells implies that both the leading and lagging template strands can form hairpins during unperturbed replication 35
. ZFN excision of a hairpin is predicted to generate gapped linear DNA. In our assay, excision of nascent strand hairpins would not be detectable after gap filling, whereas excision of template strand hairpins would result in contractions. Therefore, the contractions induced by either ZFNCTG
treatment of (CTG)102
cells (and (CTG)45
cells) support the conclusion that both the leading and lagging template strands can form hairpins, and it is also plausible that the ZFNs cleave leading strand nascent DNA hairpins. Furthermore, since instability is not detected in early passage cells in the absence of ZFN treatment, we presume that hairpin structures do not ordinarily persist long enough to be fixed as repeat length changes by a second round of replication.
Shishkin et al. have recently proposed a template switching mechanism for (GAA)•(TTC) expansion that predicts nascent strand hairpin formation as a consequence of leading strand polymerase copying of Okazaki fragments 44
. While the strong enhancement of expansions in (CAG)102
cells treated with emetine might seem at odds with this model, it is possible that these expansions occur as the (CAG)102
cells recover from the drug treatment. It has also been proposed that a common property of expandable repeats is their tendency to form noncanonical DNA structures, although the structures formed by different repeats may vary 9,45–47
. Hence, different repeats such as (GAA)•(TTC) 44
or (ATTCT)•(AGAAT) 22
may expand through structural intermediates that reflect their particular physical tendencies.
In the yeast model of large (GAA)•(TTC) expansion, instability is not dependent on fork stalling 44
. However, replication inhibition by aphidicolin, emetine, or Fen1 siRNA may potentiate expansion or contraction if the replication fork is destabilized. The present system offers several opportunities to test the contribution of the replication fork stabilization complex 48
, helicases and recombination proteins to TNR stability in human chromosomes.