Absence of MutSβ leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks

Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats.     

(CAG)*(CTG) repeats associated with neurodegenerative diseases are stable in the Escherichia coli chromosome

(CAG)(n)*(CTG)(n) expansion is associated with many neurodegenerative diseases. Repeat instability has been extensively studied in bacterial plasmids, where repeats undergo deletion at high rates. We report an assay for (CAG)(n)*(CTG)(n) deletion from the chloramphenicol acetyltransferase gene integrated into the Escherichia coli chromosome. In strain AB1157, deletion rates for 25-60 (CAG) x (CTG) repeats integrated in the chromosome ranged from 6.88 x 10(-9) to 1.33 x 10(-10), or approximately 6,300 to 660,000-fold lower than in plasmid pBR325. In contrast to the situation in plasmids, deletions occur at a higher rate when (CTG)(43), rather than (CAG)(43), comprised the leading template strand, and complete rather than partial deletions were the predominant mutation observed. Repeats were also stable on long term growth following multiple passages through exponential and stationary phase. Mutations in priA and recG increased or decreased deletion rates, but repeats were still greatly stabilized in the chromosome. The remarkable stability of (CAG)(n) x (CTG)(n) repeats in the E. coli chromosome may result from the differences in the mechanisms for replication or the probability for recombination afforded by a high plasmid copy number. The integration of (CAG)(n) x (CTG)(n) repeats into the chromosome provides a model system in which the inherent stability of these repeats reflects that in the human genome more closely.     

Slipped (CTG)*(CAG) repeats can be correctly repaired, escape repair or undergo error-prone repair

Expansion of (CTG)*(CAG) repeats, the cause of 14 or more diseases, is presumed to arise through escaped repair of slipped DNAs. We report the fidelity of slipped-DNA repair using human cell extracts and DNAs with slip-outs of (CAG)(20) or (CTG)(20). Three outcomes occurred: correct repair, escaped repair and error-prone repair. The choice of repair path depended on nick location and slip-out composition (CAG or CTG). A new form of error-prone repair was detected whereby excess repeats were incompletely excised, constituting a previously unknown path to generate expansions but not deletions. Neuron-like cell extracts yielded each of the three repair outcomes, supporting a role for these processes in (CTG)*(CAG) instability in patient post-mitotic brain cells. Mismatch repair (MMR) and nucleotide excision repair (NER) proteins hMSH2, hMSH3, hMLH1, XPF, XPG or polymerase beta were not required-indicating that their role in instability may precede that of slip-out processing. Differential processing of slipped repeats may explain the differences in mutation patterns between various disease loci or tissues.     

The effect of DNA replication mutations on CAG tract stability in yeast.

CAG repeat tracts are unstable in yeast, leading to frequent contractions and infrequent expansions in repeat tract length. To compare CAG repeats to other simple repeats and palindromic sequences, we examined the effect of DNA replication mutations, including alleles of pol alpha, pol delta, pol epsilon, and PCNA (proliferating cell nuclear antigen), on tract stability. Among the polymerase mutations, the pol delta mutation (pol3-14) destabilizes tracts with either CAG or CTG as the lagging strand template. One pol alpha mutation, pol1-1, destabilizes the orientation with CAG as the lagging strand template, but it has little effect on the CTG orientation. In contrast, the pol1-17 mutation has no effect on either orientation. Similarly, mutations in the proofreading functions of pol delta and pol epsilon, as well as a temperature-sensitive pol epsilon mutation, pol2-18, have no effect on tract stability. Three PCNA mutations, pol30-52, pol30-79, and pol30-90, all have drastic effects on tract stability. Of the three, pol30-52 is unique in yielding small tract changes that are indicative of an impairment in mismatch repair. These results show that while CAG repeats are destabilized by many of the same mutations that destabilize other simple repeats, they also have some behaviors that are suggestive of their potential to form hairpin structures.     

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.     

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.     

Long CTG.CAG repeat sequences markedly stimulate intramolecular recombination

Previous studies have shown that homologous recombination is a powerful mechanism for generation of massive instabilities of the myotonic dystrophy CTG.CAG sequences. However, the frequency of recombination between the CTG.CAG tracts has not been studied. Here we performed a systematic study on the frequency of recombination between these sequences using a genetic assay based on an intramolecular plasmid system in Escherichia coli. The rate of intramolecular recombination between long CTG.CAG tracts oriented as direct repeats was extraordinarily high; recombinants were found with a frequency exceeding 12%. Recombination occurred in both RecA(+) and RecA(-) cells but was approximately 2-11 times higher in the recombination proficient strain. Long CTG.CAG tracts recombined approximately 10 times more efficiently than non-repeating control sequences of similar length. The recombination frequency was 60-fold higher for a pair of (CTG.CAG)(165) tracts compared with a pair of (CTG.CAG)(17) sequences. The CTG.CAG sequences in orientation II (CTG repeats present on a lagging strand template) recombine approximately 2-4 times more efficiently than tracts of identical length in the opposite orientation relative to the origin of replication. This orientation effect implies the involvement of DNA replication in the intramolecular recombination between CTG.CAG sequences. Thus, long CTG.CAG tracts are hot spots for genetic recombination.     

The impact of lagging strand replication mutations on the stability of CAG repeat tracts in yeast

We have examined the stability of long tracts of CAG repeats in yeast mutants defective in enzymes suspected to be involved in lagging strand replication. Alleles of DNA ligase (cdc9-1 and cdc9-2) destabilize CAG tracts in the stable tract orientation, i.e., when CAG serves as the lagging strand template. In this orientation nearly two-thirds of the events recorded in the cdc9-1 mutant were tract expansions. While neither DNA ligase allele significantly increases the frequency of tract-length changes in the unstable orientation, the cdc9-1 mutant produced a significant number of expansions in tracts of this orientation. A mutation in primase (pri2-1) destabilizes tracts in both the stable and the unstable orientations. Mutations in a DNA helicase/deoxyribonuclease (dna2-1) or in two RNase H activities (rnh1Delta and rnh35Delta) do not have a significant effect on CAG repeat tract stability. We interpret our results in terms of the steps of replication that are likely to lead to expansion and to contraction of CAG repeat tracts.     

Hairpin structure-forming propensity of the (CCTG.CAGG) tetranucleotide repeats contributes to the genetic instability associated with myotonic dystrophy type 2

The genetic instabilities of (CCTG.CAGG)(n) tetranucleotide repeats were investigated to evaluate the molecular mechanisms responsible for the massive expansions found in myotonic dystrophy type 2 (DM2) patients. DM2 is caused by an expansion of the repeat from the normal allele of 26 to as many as 11,000 repeats. Genetic expansions and deletions were monitored in an African green monkey kidney cell culture system (COS-7 cells) as a function of the length (30, 114, or 200 repeats), orientation, or proximity of the repeat tracts to the origin (SV40) of replication. As found for CTG.CAG repeats related to DM1, the instabilities were greater for the longer tetranucleotide repeat tracts. Also, the expansions and deletions predominated when cloned in orientation II (CAGG on the leading strand template) rather than I and when cloned proximal rather than distal to the replication origin. Biochemical studies on synthetic d(CAGG)(26) and d(CCTG)(26) as models of unpaired regions of the replication fork revealed that d(CAGG)(26) has a marked propensity to adopt a defined base paired hairpin structure, whereas the complementary d(CCTG)(26) lacks this capacity. The effect of orientation described above differs from all previous results with three triplet repeat sequences (including CTG.CAG), which are also involved in the etiologies of other hereditary neurological diseases. However, similar to the triplet repeat sequences, the ability of one of the two strands to form a more stable folded structure, in our case the CAGG strand, explains this unorthodox "reversed" behavior.     

Role of replication and CpG methylation in fragile X syndrome CGG deletions in primate cells

Instability of the fragile X CGG repeat involves both maternally derived expansions and deletions in the gametes of full-mutation males. It has also been suggested that the absence of aberrant CpG methylation may enhance repeat deletions through an unknown process. The effect of CGG tract length, DNA replication direction, location of replication initiation, and CpG methylation upon CGG stability were investigated using an SV40 primate replication system. Replication-dependant deletions with 53 CGG repeats were observed when replication was initiated proximal to the repeat, with CGG as the lagging-strand template. When we initiated replication further from the repeat, while maintaining CGG as the lagging-strand template or using CCG as the lagging-strand template, significant instability was not observed. CpG methylation of the unstable template stabilized the repeat, decreasing both the frequency and the magnitude of deletion events. Furthermore, CpG methylation slowed the efficiency of replication for all templates. Interestingly, replication forks displayed no evidence of a block at the CGG repeat tract, regardless of replication direction or CpG methylation status. Templates with 20 CGG repeats were stable under all circumstances. These results reveal that CGG deletions occur during replication and are sensitive to replication-fork dynamics, tract length, and CpG methylation.     

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.     

DnaB helicase affects the initiation specificity of Escherichia coli primase on single-stranded DNA templates

The effect of DnaB helicase on the initiation specificity of primase was studied biochemically using a series of single-stranded DNA templates in which each nucleotide of the trinucleotide d(CTG) initiation sequence was systematically varied. DnaB helicase accelerated the rate of primer syntheisis, prevented "overlong" primers from forming and decreased the initiation specificity of primase. In the presence of DnaB helicase, all trinucleotides could serve as the primer initiation site although there was a distinct preference for d(CAG). These data may explain the high chromosomal prevalence of octanucleotides containing CTG on the leading strand and its complement CAG on the lagging strand. The specificity of DnaB helicase places it on the lagging strand template where it stimulates the initiation of Okazaki fragment synthesis. In the absence of DnaB helicase, primase preferentially primed the d(CTG) template. In the presence of DnaB helicase, the initiation preference was not only altered but also the preferred initiating nucleotide was found to be GTP rather than ATP, for both the d(CTG) and the d(CAG) templates. This suggested that the specificity of primase for the d(CTG) initiation trinucleotide was predominantly unaffected in the absence of DnaB helicase on short ssDNA templates, whereas in conjunction with DnaB helicase, the specificity was altered and this alteration has significant implications in the replication of Escherichia coli chromosome in vivo.     

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.     

Deletion errors generated during replication of CAG repeats.

Triplet repeat sequence instability is associated with hereditary neurological diseases and with certain types of cancer. Here we study one form of this instability, deletion of triplet repeats during replication of template (CAG)(n)sequences by DNA polymerases. To monitor loss of triplet codons, we inserted (CAG)(9)and (CAG)(17)repeats into the lacZ sequence in M13mp2 and changed one repeat to a TAG codon to yield DNA substrates with colorless plaque phenotypes. Templates containing these inserts within gaps were copied and errors were scored as blue plaque Lac revertants whose DNA was sequenced to determine if loss of the TAG codon resulted from substitutions or deletions. DNA synthesis by either DNA polymerase beta or exonuclease-deficient T7 DNA polymerase produced deletions involving loss of from 1 to 8 of 9 or 15 of 17 repeats. Thus, these polymerases utilize misaligned template-primers containing from 3 to 45 extra template strand nucleotides. Deletion frequencies were much higher than substitution frequencies at the TAG codon in certain repeats, indicating that triplet repeats are at high risk for mutation in the absence of error correction. Proofreading-proficient T7 DNA polymerase generated deletions at 2- to 10-fold lower frequencies than did its exonuclease-deficient derivative. This suggests that misaligned triplet repeat sequences are subject to proofreading, but at reduced efficiency compared to editing of single-base mismatches.     

DNA double-strand breaks induce deletion of CTG.CAG repeats in an orientation-dependent manner in Escherichia coli

The influences of double-strand breaks (DSBs) within a triplet repeat sequence on its genetic instabilities (expansions and deletions) related to hereditary neurological diseases was investigated. Plasmids containing 43 or 70 CTG.CAG repeats or 43 CGG.CCG repeats were linearized in vitro near the center of the repeats and were transformed into parental, RecA-dependent homologous recombination-deficient, or RecBC exonuclease-deficient Escherichia coli. The resulting repair process considerably increased deletion of the repeating sequence compared to the circular DNA controls. Unexpectedly, the orientation of the insert relative to the unidirectional ColE1 origin of replication affected the amount of instability generated during the repair of the DSB. When the CTG strand was the template for lagging-strand synthesis, instability was increased, most markedly in the recA- strain. Results indicated that RecA and/or RecBC might play a role in DSB repair within the triplet repeat. Altering the length, orientation, and sequence composition of the triplet repeat suggested an important role of DNA secondary structures during repair intermediates. Hence, we hypothesize that ColE1 origin-dependent replication was involved during the repair of the DSB. A model is presented to explain the mechanisms of the observed genetic instabilities.     

The Werner Syndrome Protein Promotes CAG/CTG Repeat Stability by Resolving Large (CAG)n/(CTG)n Hairpins

Expansion of CAG/CTG repeats causes certain neurological and neurodegenerative disorders, and the formation and subsequent persistence of stable DNA hairpins within these repeats are believed to contribute to CAG/CTG repeat instability. Human cells possess a DNA hairpin repair (HPR) pathway, which removes various (CAG)(n) and (CTG)(n) hairpins in a nick-directed and strand-specific manner. Interestingly, this HPR system processes a (CTG)(n) hairpin on the template DNA strand much less efficiently than a (CAG)(n) hairpin on the same strand (Hou, C., Chan, N. L., Gu, L., and Li, G. M. (2009) Incision-dependent and error-free repair of (CAG)(n)/(CTG)(n) hairpins in human cell extracts. Nat. Struct. Mol. Biol. 16, 869-875), suggesting the involvement of an additional component for (CTG)(n) HPR. To identify this activity, a functional in vitro HPR assay was used to screen partially purified HeLa nuclear fractions for their ability to stimulate (CTG)(n) HPR. We demonstrate here that the stimulating activity is the Werner syndrome protein (WRN). Although WRN contains both a 3'→5' helicase activity and a 3'→5' exonuclease activity, the stimulating activity was found to be the helicase activity, as a WRN helicase mutant failed to enhance (CTG)(n) HPR. Consistently, WRN efficiently unwound large (CTG)(n) hairpins and promoted DNA polymerase δ-catalyzed DNA synthesis using a (CTG)(n) hairpin as a template. We, therefore, conclude that WRN stimulates (CTG)(n) HPR on the template DNA strand by resolving the hairpin so that it can be efficiently used as a template for repair or replicative synthesis.     

Replication restart: a pathway for (CTG).(CAG) repeat deletion in Escherichia coli

(CTG)n.(CAG)n repeats undergo deletion at a high rate in plasmids in Escherichia coli in a process that involves RecA and RecB. In addition, DNA replication fork progression can be blocked during synthesis of (CTG)n.(CAG)n repeats. Replication forks stalled at (CTG)n.(CAG)n repeats may be rescued by replication restart that involves recombination as well as enzymes involved in replication and DNA repair, and this process may be responsible for the high rate of repeat deletion in E. coli. To test this hypothesis (CAG)n.(CTG)n deletion rates were measured in several E. coli strains carrying mutations involved in replication restart. (CAG)n.(CTG)n deletion rates were decreased, relative to the rates in wild type cells, in strains containing mutations in priA, recG, ruvAB, and recO. Mutations in priB and priC resulted in small reductions in deletion rates. In a recF strain, rates were decreased when (CAG)n comprised the leading template strand, but rates were increased when (CTG)n comprised the leading template. Deletion rates were increased slightly in a recJ strain. The mutational spectra for most mutant strains were altered relative to those in parental strains. In addition, purified PriA and RecG proteins showed unexpected binding to single-stranded, duplex, and forked DNAs containing (CAG)n and/or (CTG)n loop-outs in various positions. The results presented are consistent with an interpretation that the high rates of trinucleotide repeat instability observed in E. coli result from the attempted restart of replication forks stalled at (CAG)n.(CTG)n repeats.     

Instability of CTG Repeats is Governed by the Position of a DNA Base Lesion through Base Excision Repair

Trinucleotide repeat (TNR) expansions and deletions are associated with human neurodegeneration and cancer. However, their underlying mechanisms remain to be elucidated. Recent studies have demonstrated that CAG repeat expansions can be initiated by oxidative DNA base damage and fulfilled by base excision repair (BER), suggesting active roles for oxidative DNA damage and BER in TNR instability. Here, we provide the first evidence that oxidative DNA damage can induce CTG repeat deletions along with limited expansions in human cells. Biochemical characterization of BER in the context of (CTG)₂₀ repeats further revealed that repeat instability correlated with the position of a base lesion in the repeat tract. A lesion located at the 5′-end of CTG repeats resulted in expansion, whereas a lesion located either in the middle or the 3′-end of the repeats led to deletions only. The positioning effects appeared to be determined by the formation of hairpins at various locations on the template and the damaged strands that were bypassed by DNA polymerase β and processed by flap endonuclease 1 with different efficiency. Our study indicates that the position of a DNA base lesion governs whether TNR is expanded or deleted through BER.     

Mutations in yeast replication proteins that increase CAG/CTG expansions also increase repeat fragility

Expansion of trinucleotide repeats (TNRs) is the causative mutation in several human genetic diseases. Expanded TNR tracts are both unstable (changing in length) and fragile (displaying an increased propensity to break). We have investigated the relationship between fidelity of lagging-strand replication and both stability and fragility of TNRs. We devised a new yeast artificial chromomosme (YAC)-based assay for chromosome breakage to analyze fragility of CAG/CTG tracts in mutants deficient for proteins involved in lagging-strand replication: Fen1/Rad27, an endo/exonuclease involved in Okazaki fragment maturation, the nuclease/helicase Dna2, RNase HI, DNA ligase, polymerase delta, and primase. We found that deletion of RAD27 caused a large increase in breakage of short and long CAG/CTG tracts, and defects in DNA ligase and primase increased breakage of long tracts. We also found a correlation between mutations that increase CAG/CTG tract breakage and those that increase repeat expansion. These results suggest that processes that generate strand breaks, such as faulty Okazaki fragment processing or DNA repair, are an important source of TNR expansions.