Binding sites for the ORC and minichromosome maintenance complex have been identified at several metazoan replication origins (2
); however, these sites are not sufficient to specify a chromosomal region as an origin of replication in the absence of a DUE (14
). Not surprisingly, local regions of predicted helically unstable DNA are common features of mammalian replication origins, including the c-myc
replication origin (18
). The replication origin activity associated with the easily unwound DNA elements at the mutant SCA10 locus and at the engineered ectopic c-myc
replicator argue that, in addition to possible roles in the modification of chromatin structure or protein binding, DUEs facilitate the supercoiling-induced helix unwinding of the DNA template strands during replication initiation.
We have previously shown that (ATTCT)n
repeat tracts longer than those in normal human alleles act as DUEs and support aberrant DNA replication initiation in vitro (46
). These observations led to the hypothesis that expanded repeats may promote aberrant replication in human chromosomes and cause the instability of repeats for which disease is associated with increased repeat length. Here, we present data confirming these hypotheses. First, we show that the endogenous (ATTCT)n
tract within the ataxin 10 gene in normal cells shows only background replication origin activity but that origin activity is elevated at least 5- to 10-fold in lymphoblasts from SCA10 patients with expanded (ATTCT)n
tracts. Second, we show that the (ATTCT)n
tracts for which n
is 27 or 48 function as DUEs replacing the natural DUE within the c-myc
replicator, which is essential for replication initiation. Repeat tracts where n
is 8 or 13, that is, below or within the normal range of 10 to 22 repeats, fail to support DNA replication initiation. Third, we show that (ATTCT)48
at the ectopic location but in the absence of c-myc
replicator sequences does not support replication. Fourth, we show that longer-than-normal repeat tracts undergo two- to fourfold expansion, from 48 to ~170 and from 27 to ~125 repeats, during growth in human cells. The expansion, significantly, is dependent on the proximity of replication origin activity; expansion is not observed during replication from a distant origin.
tracts longer than the normal range are unstable when placed close to sites of replication initiation but appear to be stable when replicated from distal origins. The simplest explanation for expansion of the AT-rich pentanucleotide tracts between preserved ATX10 flanking sequences is replication slippage (51
) in which greater phasing of Okazaki fragment initiation sites proximal to the origin favors the formation of metastable loops in the newly synthesized strand (11
). Instability may also be a direct consequence of replication if (ATTCT)n
sequences are unwound and recombinogenic when they constitute the majority of a newly synthesized strand near an origin but are stable when they comprise the 3′ end of a long nascent strand replicated from a distal origin (46
). Local amplification of the ATX10 DUEs in the Δ5S27 and Δ5S48 cell lines would be consistent with such mechanisms. Alternatively, the length- and position-dependent instability of (ATTCT)n
tracts may not be related to replication per se but could reflect a chromosome structure permissive for DNA unwinding and recombination at the origin or changes in the protein composition of replication forks as they progress.
In the absence of the c-myc replicator, the ectopic FRT site is replicated primarily from a downstream origin (unpublished results). Thus, we cannot formally rule out the possibility that a change in replication polarity is responsible for the stability of the (ATTCT)n tracts in the absence of the proximal c-myc replicator. However, in similar experiments, (CAG)102 tracts are unstable irrespective of orientation when flanked by the ectopic c-myc replicator but stable in the absence of a proximal origin (G. Liu, unpublished results).
After ~250 population doublings spPCRs of Δ5S27 and Δ5S48 cells show similar patterns of (ATTCT)n tract amplification, which we interpret to indicate expansion by a similar mechanism in many cells of the population. While it cannot be totally excluded that several subpopulations with favored repeat lengths have overtaken the culture, the similarity in the offset patterns from the independently derived Δ5S27 and Δ5S48 cells argues strongly that the distributions of amplified repeat lengths reflect the mechanism of expansion rather than mitotic drive.
The present data indicate that the expansion of (ATTCT)n
tracts that leads to SCA10 causes abnormal replication origin activity and genomic instability. These results suggest a model in which sporadic replication origin activity at the ATX10 locus promotes increases in (ATTCT)n
repeat length, which potentiate origin activity and the formation of larger tracts (46
). The absence of origin activity at the X chromosome (ATTCT)38
tract or the (ATTCT)8
repeats at the ectopic locus in the absence of c-myc
replicator sequences implies that structures in addition to a DUE are required to specify a chromosomal origin, although it has not been possible to test whether highly extended, disease-length (ATTCT)n
microsatellites by themselves are sufficient for origin activity. Nevertheless, a requirement for multiple replicator elements is consistent with previous results (14
). Thus, the inefficient origin activity of the wild-type ATX10 locus may reflect the absence of appropriate ancillary elements or epigenetic structure. That expansion of the (ATTCT)n
tract is sufficient to enable origin activity suggests that a change in DNA or chromatin structure is of primary importance. The sequence specificity of metazoan ORC binding is modest compared to its preference for binding to supercoiled DNA (48
). The demonstration that origin activity is low at the wild-type ATX10 locus but significantly elevated at the expanded ATX10 locus raises the question of whether prereplicative complex proteins are bound but inactive at the wild-type ATX10 locus or are recruited to the ATX10 locus as a result of expansion of the (ATTCT)n
tract and a concomitant alteration of DNA topology and chromosome organization. Experiments are currently under way to address these questions.
These data present strong evidence validating models suggesting that expanded DNA repeats promote aberrant DNA replication initiation (33
) and that the activation of cryptic origins leads to genomic instability (12
). Moreover, they suggest a molecular mechanism associated with (ATTCT)n
repeat expansion: the initiation of DNA replication. In addition, the results suggest that the analysis of repeat instability in this ectopic location in HeLa cells provides a valuable model for studying cis
- and trans
-acting factors affecting repeat instability.