Weak strand displacement activity enables human DNA polymerase beta to expand CAG/CTG triplet repeats at strand breaks

Using synthetic DNA constructs in vitro, we find that human DNA polymerase beta effectively catalyzes CAG/CTG triplet repeat expansions by slippage initiated at nicks or 1-base gaps within short (14 triplet) repeat tracts in DNA duplexes under physiological conditions. In the same constructs, Escherichia coli DNA polymerase I Klenow Fragment exo(-) is much less effective in expanding repeats, because its much stronger strand displacement activity inhibits slippage by enabling rapid extension through two downstream repeats into flanking non-repeat sequence. Polymerase beta expansions of CAG/CTG repeats, observed over a 32-min period at rates of approximately 1 triplet added per min, reveal significant effects of break type (nick versus gap), strand composition (CTG versus CAG), and dNTP substrate concentration, on repeat expansions at strand breaks. At physiological substrate concentrations (1-10 microm of each dNTP), polymerase beta expands triplet repeats with the help of weak strand displacement limited to the two downstream triplet repeats in our constructs. Such weak strand displacement activity in DNA repair at strand breaks may enable short tracts of repeats to be converted into longer, increasingly mutable ones associated with neurological diseases.     

Generation of long tracts of disease-associated DNA repeats

The generation of long uninterrupted DNA repeats is important for the study of repeat instability associated with several human genetic diseases, including myotonic dystrophy type 1. However, obtaining defined lengths of long repeats in vitro has been problematic. Strand slippage and/or DNA secondary structure formation may prevent efficient ligation. For example, a purified (CTG)140.(CAG)140 repeat fragment containing 4-bp AGCA/TGCT overhanging ends ligated poorly using T4 or Escherichia coli DNA ligase, although limited repeat ligation occurred using thermostable DNA ligase. Here we describe a general procedure for ligating multimers of DNA repeats. Multimers are efficiently ligated when slippage is prevented or when DNA repeats contain a single G/C overhang. A cloning vector is designed from which pure repeat fragments containing a G/C overhang can be generated for further ligation. (CAG)n.(CTG)n DNA molecules longer than 800 bp were generated using this approach. This approach also worked for (GAA)n.(TTC)n, (CCTG)n-(CAGG)n, and (ATTCT)n.(AGAAT)n tracts associated with Friedreich ataxia, DM2, and spinocerebellar ataxia type 10, respectively.     

Chemotherapeutically induced deletion of expanded triplet repeats

The number of neurodegenerative disorders associated with the expansion of DNA repeats, currently about 18, continues to increase as additional diseases caused by this novel type of mutation are identified. Typically, expanded repeats are biased toward further expansion upon intergenerational transmission, and disease symptoms show an earlier age of onset and greater severity as the length of the triplet repeat tract increases. Most diseases exhibit progressive neurological and/or muscular degeneration that can lead to total disability and death. As yet, no treatment exists for the genetic basis of any repeat disease. Given that the severity of these diseases is related to repeat tract length, reducing repeat lengths might delay the onset and reduce disease severity. Here, we test the hypothesis that the introduction of damage into DNA, which results in subsequent repair events, can lead to an increased rate of repeat deletion. Applying a sensitive genetic assay in Escherichia coli [Mut. Res. 502 (2002) 25], we demonstrate that certain DNA damaging agents, including EMS, ENU, UV light, and anticancer agents mitomycin C, cisplatin, and X-rays increase the rate of deletion of (CTG).(CAG) repeats in a length and orientation dependent fashion. In addition, oxidative damage to DNA also increases the deletion rate of repeats. These results suggest that a chemotherapeutic approach to the reduction in triplet repeat length may provide one possible rationale to slow, stop, or reverse the progression of these diseases.     

TempliPhi,phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing

We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification. Large amounts of product (1-3 microg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture. Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed.     

Isolation of CAG/CTG repeats from within the chromosome 2p21-p24 locus for autosomal dominant spastic paraplegia (SPG4) by YAC fragmentation

Pure autosomal dominant spastic paraplegia (SPG) is a genetically heterogeneous neurodegenerative disorder of the central nervous system clinically characterized by progressive spasticity mainly affecting the lower limbs. Three distinct loci have been mapped to chromosomes 14q (SPG3), 2p (SPG4) and 15q (SPG6). In particular, SPG4 families show striking intrafamilial variability suggestive of anticipation and evidence has been provided that CAG/CTG repeat expansions may be involved. To isolate CAG/CTG repeat containing sequences from within the SPG4 candidate region, a novel approach was developed. Fragmentation vectors were assembled allowing direct fragmentation of yeast artificial chromosomes (YACs) with a short (> or = 21 bp) CAG/CTG sequence as the target site for homologous recombination. We used the CAG/CTG YAC fragmentation vectors to isolate CAG/CTG containing sequences from four YACs spanning the SPG4 candidate region between D2S400 and D2S367. A total of four CAG/CTG containing sequences were isolated of which three were novel. However, none of the four CAG/CTG repeats showed expanded alleles in two Belgian SPG4 families. In addition, we showed that the CAG/CTG alleles detected by the repeat expansion detection (RED) method could be fully explained by two polymorphic nonpathogenic CAG/CTG repeats on chromosomes 17 and 18, respectively. Also, the RED expansions in six SPG families could not be explained by amplification of the CAG/CTG repeats at the SPG4 locus. Together, our data do not support the hypothesis of a CAG/CTG repeat expansion as the molecular mechanism underlying SPG4 pathology.     

In vitro repair of DNA hairpins containing various numbers of CAG/CTG trinucleotide repeats

Expansion of CAG/CTG trinucleotide repeats (TNRs) in humans is associated with a number of neurological and neurodegenerative disorders including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism leads to TNR instability. However, the molecular mechanism by which cells recognize and repair CAG/CTG hairpins is largely unknown. Recent studies have identified a novel DNA repair pathway specifically removing (CAG)(n)/(CTG)(n) hairpins, which is considered a major mechanism responsible for TNR instability. The hairpin repair (HPR) system targets the repeat tracts for incisions in the nicked strand in an error-free manner. To determine the substrate spectrum of the HPR system and its ability to process smaller hairpins, which may be the intermediates for CAG/CTG expansions, we constructed a series of CAG/CTG hairpin heteroduplexes containing different numbers of repeats (from 5 to 25) and examined their repair in human nuclear extracts. We show here that although repair efficiencies differ slightly among these substrates, removal of the individual hairpin structures all involve endonucleolytic incisions within the repeat tracts in the nicked DNA strand. Analysis of the repair intermediates defined specific incision sites for each substrate, which were all located within the repeat regions. Mismatch repair proteins are not required for, nor do they inhibit, the processing of smaller hairpin structures. These results suggest that the HPR system ensures CAG/CTG stability primarily by removing various sizes of (CAG)(n)/(CTG)(n) hairpin structures during DNA metabolism.     

Replication inhibitors modulate instability of an expanded trinucleotide repeat at the myotonic dystrophy type 1 disease locus in human cells

Gene-specific CTG/CAG repeat expansion is associated with at least 14 human diseases, including myotonic dystrophy type 1 (DM1). Most of our understanding of trinucleotide instability is from nonhuman models, which have presented mixed results, supporting replication errors or processes independent of cell division as causes. Nevertheless, the mechanism occurring at the disease loci in patient cells is poorly understood. Using primary fibroblasts derived from a fetus with DM1, we have shown that spontaneous expansion of the diseased (CTG)(216) allele occurred in proliferating cells but not in quiescent cells. Expansions were "synchronous," with mutation frequencies approaching 100%. Furthermore, cells were treated with agents known to alter DNA synthesis but not to directly damage DNA. Inhibiting replication initiation with mimosine had no effect upon instability. Inhibiting both leading- and lagging-strand synthesis with aphidicolin or blocking only lagging strand synthesis with emetine significantly enhanced CTG expansions. It was striking that only the expanded DM1 allele was altered, leaving the normal allele, (CTG)(12), and other repeat loci unaffected. Standard and small-pool polymerase chain reaction revealed that inhibitors enhanced the magnitude of short expansions in most cells threefold, whereas 11%-25% of cells experienced gains of 122-170 repeats, to sizes of (CTG)(338)-(CTG)(386). Similar results were observed for an adult DM1 cell line. Our results support a role for the perturbation of replication fork dynamics in DM1 CTG expansions within patient fibroblasts. This is the first report that repeat-length alterations specific to a disease allele can be modulated by exogenously added compounds.     

Chemical modifiers of unstable expanded simple sequence repeats: what goes up, could come down

A mounting number of inherited human disorders, including Huntington disease, myotonic dystrophy, fragile X syndrome, Friedreich ataxia and several spinocerebellar ataxias, have been associated with the expansion of unstable simple sequence DNA repeats. Despite a similar genetic basis, pathogenesis in these disorders is mediated by a variety of both loss and gain of function pathways. Thus, therapies targeted at downstream pathology are likely to be disease specific. Characteristically, disease-associated expanded alleles in these disorders are highly unstable in the germline and somatic cells, with a tendency towards further expansion. Whereas germline expansion accounts for the phenomenon of anticipation, tissue-specific, age-dependent somatic expansion may contribute towards the tissue-specificity and progressive nature of the symptoms. Thus, somatic expansion presents as a novel therapeutic target in these disorders. Suppression of somatic expansion should be therapeutically beneficial, whilst reductions in repeat length could be curative. It is well established that both cis- and trans-acting genetic modifiers play key roles in the control of repeat dynamics. Importantly, recent data have revealed that expanded CAG.CTG repeats are also sensitive to a variety of trans-acting chemical modifiers. These data provide an exciting proof of principle that drug induced suppression of somatic expansion might indeed be feasible. Moreover, as our understanding of the mechanism of expansion is refined more rational approaches to chemical intervention in the expansion pathway can be envisioned. For instance, the demonstration that expansion of CAG.CTG repeats is dependent on the Msh2, Msh3 and Pms2 genes, highlights components of the DNA mismatch repair pathway as therapeutic targets. In addition to potential therapeutic applications, the response of expanded simple repeats to genotoxic assault suggests such sequences could also have utility as bio-monitors of environmentally induced genetic damage in the soma.     

58 Packaging trinucleotide repeats: the effect of CAG/CTG repeats on nucleosome formation

In neurological diseases such as fragile X syndrome, spinal and bulbar muscular atrophy, myotonic dystrophy, and Huntington’s disease, the molecular basis of pathogenicity is the presence of an expanded trinucleotide repeat (TNR) tract (Ashley & Warren, 1995). TNRs implicated in many of these diseases are composed of CAG/CTG repeats. For example, in healthy individuals 5–35, CAG/CTG TNR repeats are present in the huntingtin gene. However, individuals with 40 or greater repeats will develop Huntington’s disease (Andrew et al., 1993). We are particularly interested in how these TNR sequences are packaged in chromatin. Recent evaluations of CAG/CTG TNR sequences in our laboratory have demonstrated that the repeats increase the propensity for the DNA sequences to incorporate into nucleosomes, where nucleosomes represent the minimal unit of packaging in chromatin (Volle & Delaney, 2012). In this work, we are interested in determining the minimum number of CAG/CTG repeats required to confer a significant increase in nucleosome incorporation relative to sequences that lack the TNR sequence. By defining the changes imposed on these fundamental interactions by the presence of a CAG/CTG repeat tract, we will gain insight into the possible interactions that allow for the expansion of these TNR tracts.     

Replication and expansion of trinucleotide repeats in yeast

The mechanisms of trinucleotide repeat expansions, underlying more than a dozen hereditary neurological disorders, are yet to be understood. Here we looked at the replication of (CGG)(n) x (CCG)(n) and (CAG)(n) x (CTG)(n) repeats and their propensity to expand in Saccharomyces cerevisiae. Using electrophoretic analysis of replication intermediates, we found that (CGG)(n) x (CCG)(n) repeats significantly attenuate replication fork progression. Replication inhibition for this sequence becomes evident at as few as approximately 10 repeats and reaches a maximal level at 30 to 40 repeats. This is the first direct demonstration of replication attenuation by a triplet repeat in a eukaryotic system in vivo. For (CAG)(n) x (CTG)(n) repeats, on the contrary, there is only a marginal replication inhibition even at 80 repeats. The propensity of trinucleotide repeats to expand was evaluated in a parallel genetic study. In wild-type cells, expansions of (CGG)(25) x (CCG)(25) and (CAG)(25) x (CTG)(25) repeat tracts occurred with similar low rates. A mutation in the large subunit of the replicative replication factor C complex (rfc1-1) increased the expansion rate for the (CGG)(25) repeat approximately 50-fold but had a much smaller effect on the expansion of the (CTG)(25) repeat. These data show dramatic sequence-specific expansion effects due to a mutation in the lagging strand DNA synthesis machinery. Together, the results of this study suggest that expansions are likely to result when the replication fork attempts to escape from the stall site.