Role of recombination and replication fork restart in repeat instability

Eukaryotic genomes contain many repetitive DNA sequences that exhibit size instability. Some repeat elements have the added complication of being able to form secondary structures, such as hairpin loops, slipped DNA, triplex DNA or G-quadruplexes. Especially when repeat sequences are long, these DNA structures can form a significant impediment to DNA replication and repair, leading to DNA nicks, gaps, and breaks. In turn, repair or replication fork restart attempts within the repeat DNA can lead to addition or removal of repeat elements, which can sometimes lead to disease. One important DNA repair mechanism to maintain genomic integrity is recombination. Though early studies dismissed recombination as a mechanism driving repeat expansion and instability, recent results indicate that mitotic recombination is a key pathway operating within repetitive DNA. The action is two-fold: first, it is an important mechanism to repair nicks, gaps, breaks, or stalled forks to prevent chromosome fragility and protect cell health; second, recombination can cause repeat expansions or contractions, which can be deleterious. In this review, we summarize recent developments that illuminate the role of recombination in maintaining genome stability at DNA repeats.     

Satellite DNA and disease--possible mechanisms. Minisatellite instability

The main possible molecular mechanisms of minisatellite DNA instability are reviewed and compared with those of the trinucleotide repeat instability. Evidence indicating that some human diseases are associated with minisatellite DNA instability is presented including data on minisatellite DNA expansion.     

Most meiotic CAG repeat tract-length alterations in yeast are<Emphasis Type="Italic"> SPO11</Emphasis> dependent

The expansion of trinucleotide repeat sequences associated with hereditary neurological diseases is believed from earlier studies to be due to errors in DNA replication. However, more recent studies have indicated that recombination may play a significant role in triplet repeat expansion. CAG repeat tracts have been shown to induce double-strand breaks (DSBs) during meiosis in yeast, and DSB formation is dependent on the meiotic recombination machinery. The rate of meiotic instability is several fold higher than mitotic instability. To determine whether DSB repair is responsible for the high rate of repeat tract-length alterations, the frequencies of meiotic repeat-tract instability were compared in wild-type and spo11 mutant strains. In the spo11 background, the rate of meiotic repeat-tract instability remained at the mitotic level, suggesting that meiotic alterations of CAG repeat tracts in yeast occur by the recombination mechanism. Several of these meiotic tract-length alterations are due to DSB repair involving use of the sister chromatid as a template.     

Advances in the application of germline tandem repeat instability for in situ monitoring

Alterations in tandem repetitive DNA sequences such as minisatellite DNA and expanded simple tandem repeats (ESTRs) may provide useful biomarkers of induced germline effects. In this review, I describe the differences between ESTRs and minisatellites with respect to their structure and mutational mechanisms, and discuss field applications measuring induced germline instability. It is evident that both types of loci have high rates of mutation that facilitate the measurement of induced mutation measured in relatively small numbers of samples following environmentally relevant exposures. Several research groups have used these loci to demonstrate a significant increase in germline mutation in humans and animals exposed to radioactive or chemical pollutants in their natural environment. Mutations are manifested as gains or losses in repeat units and are detected either by pedigree screening or by PCR amplification of sperm DNA. Mutations at both ESTRs and minisatellites appear to arise via indirect mechanisms rather than by direct damage to the repeat locus itself. Most interestingly, ESTR instability following radiation has been shown to be heritable and transmitted to subsequent generations. An understanding of the mechanisms involved in induced instability is required in order to begin to decipher the potential biological implications of increased germline tandem repeat mutation. Furthermore, relatively few studies have investigated the ability of different genotoxins to induce tandem repeat instability. Such laboratory-based experiments will be crucial in clarifying the particular environmental or occupational exposures that should be targeted for future studies and for isolating and subsequently identifying the putative mutagens in complex environmental matrices.     

DNA mismatch repair in trinucleotide repeat instability

Trinucleotide repeat expansions cause over 30 severe neuromuscular and neurodegenerative disorders, including Huntington's disease, myotonic dystrophy type 1, and fragile X syndrome. Although previous studies have substantially advanced the understanding of the disease biology, many key features remain unknown. DNA mismatch repair(MMR) plays a critical role in genome maintenance by removing DNA mismatches generated during DNA replication. However, MMR components,particularly mismatch recognition protein MutSβ and its interacting factors MutLα and MutLγ, have been implicated in trinucleotide repeat instability. In this review, we will discuss the roles of these key MMR proteins in promoting trinucleotide repeat instability.     

Micronuclei and sister chromatid exchanges induced by capsaicin in human lymphocytes

Escherichia coli has provided an important model system for understanding the molecular basis for genetic instabilities associated with repeated DNA. Changes in triplet repeat length during growth following transformation in E. coli have been used as a measure of repeat instability. However, very little is known about the molecular and biological changes that may occur on transformation. Since only a small proportion of viable cells become competent, uncertainty exists regarding the nature of these transformed cells. To establish whether the process of transformation can be inherently mutagenic for certain DNA sequences, we used a genetic assay in E. coli to compare the frequency of genetic instabilities associated with transformation with those occurring in plasmid maintained in E. coli. Our results indicate that, for certain DNA sequences, bacterial transformation can be highly mutagenic. The deletion frequency of a 106 bp perfect inverted repeat is increased by as much as a factor of 2 x 10(5) following transformation. The high frequency of instability was not observed when cells stably harboring plasmid were rendered competent. Thus, the process of transformation was required to observe the instability. Instabilities of (CAG).(CTG) repeats are also dramatically elevated upon transformation. The magnitude of the instability is dependent on the nature and length of the repeat. Differences in the methylation status of plasmid used for transformation and the methylation and restriction/modification systems present in the bacterial strain used must also be considered in repeat instability measurements. Moreover, different E. coli genetic backgrounds show different levels of instability during transformation.     

Mechanisms of tandem repeat instability in bacteria

Hypermutable tandem repeat sequences (TRSs) are present in the genomes of both prokaryotic and eukaryotic organisms. Numerous studies have been conducted in several laboratories over the past decade to investigate the mechanisms responsible for expansions and contractions of microsatellites (a subset of TRSs with a repeat length of 1-6 nucleotides) in the model prokaryotic organism Escherichia coli. Both the frequency of tandem repeat instability (TRI), and the types of mutational events that arise, are markedly influenced by the DNA sequence of the repeat, the number of unit repeats, and the types of cellular pathways that process the TRS. DNA strand slippage is a general mechanism invoked to explain instability in TRSs. Misaligned DNA sequences are stabilized both by favorable base pairing of complementary sequences and by the propensity of TRSs to form relatively stable secondary structures. Several cellular processes, including replication, recombination and a variety of DNA repair pathways, have been shown to interact with such structures and influence TRI in bacteria. This paper provides an overview of our current understanding of mechanisms responsible for TRI in bacteria, with an emphasis on studies that have been carried out in E. coli. In addition, new experimental data are presented, suggesting that TLS polymerases (PolII, PolIV and PolV) do not contribute significantly to TRI in E. coli.     

Disease-associated repeat instability and mismatch repair

Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential.     

The contribution of cis-elements to disease-associated repeat instability: clinical and experimental evidence

Alterations in the length (instability) of gene-specific microsatellites and minisatellites are associated with at least 35 human diseases. This review will discuss the various cis-elements that contribute to repeat instability, primarily through examination of the most abundant disease-associated repetitive element, trinucleotide repeats. For the purpose of this review, we define cis-elements to include the sequence of the repeat units, the length and purity of the repeat tracts, the sequences flanking the repeat, as well as the surrounding epigenetic environment, including DNA methylation and chromatin structure. Gender-, tissue-, developmental- and locus-specific cis-elements in conjunction with trans-factors may facilitate instability through the processes of DNA replication, repair and/or recombination. Here we review the available human data that supports the involvement of cis-elements in repeat instability with limited reference to model systems. In diverse tissues at different developmental times and at specific loci, repetitive elements display variable levels of instability, suggesting vastly different mechanisms may be responsible for repeat instability amongst the disease loci and between various tissues.     

The influence of interstitial telomeric sequences on chromosome instability in human cells

Although most telomere repeat sequences are found at the ends of chromosomes, some telomeric repeat sequences are also found at intrachromosomal locations in mammalian cells. Several studies have found that these interstitial telomeric repeat sequences can promote chromosome instability in rodent cells, either spontaneously or following ionizing radiation. In the present study we describe the extensive cytogenetic analysis of three different human cell lines with plasmids containing telomeric repeat sequences integrated at interstitial sites. In two of these cell lines, Q18 and P8SX, instability has been detected in the chromosome containing the integrated plasmid, involving breakage/fusion/bridge cycles or amplification of the plasmid DNA, respectively. However, the data suggest that the instability observed is characteristic of the general instability in these cell lines and that the telomeric repeat sequences themselves are not responsible. Consistent with this interpretation, the chromosome containing an integrated plasmid with 500 bp of telomeric repeat sequences is highly stable in the third cell line, SNG28, which has a relatively stable genome. The stability of the chromosome containing the integrated plasmid sequences in SNG28 makes this an excellent cell line to study the effect of ionizing radiation on the stability of interstitial telomeric sequences in human cells.     

Repeat instability at human minisatellites arising from meiotic recombination.

Little is known about the role of meiotic recombination processes such as unequal crossover in driving instability at tandem repeat DNA. Methods have therefore been developed to detect meiotic crossovers within two different GC-rich minisatellite repeat arrays in humans, both in families and in sperm DNA. Both loci normally mutate in the germline by complex conversion-like transfer of repeats between alleles. Analysis shows that inter-allelic unequal crossovers also occur at both loci, although at low frequency, to yield simple recombinant repeat arrays with exchange of flanking markers. Equal crossovers between aligned alleles, resulting in recombinant alleles but without change in repeat copy number, also occur in sperm at a similar frequency to unequal crossovers. Both crossover and conversion show polarity in the repeat array and are co-suppressed in an allele showing unusual germline stability. This provides evidence that minisatellite conversion and crossover arise by a common mechanism, perhaps by alternative processing of a meiotic recombination initiation complex, and implies that minisatellite instability is a by-product of meiotic recombination in repeat DNA. While minisatellite recombination is infrequent, crossover rates indicate that the unstable end of a human minisatellite can act as a recombination warm-spot, even between sequence-heterologous alleles.     

Involvement of the nucleotide excision repair protein UvrA in instability of CAG*CTG repeat sequences in Escherichia coli.

Several human genetic diseases have been associated with the genetic instability, specifically expansion, of trinucleotide repeat sequences such as (CTG)(n).(CAG)(n). Molecular models of repeat instability imply replication slippage and the formation of loops and imperfect hairpins in single strands. Subsequently, these loops or hairpins may be recognized and processed by DNA repair systems. To evaluate the potential role of nucleotide excision repair in repeat instability, we measured the rates of repeat deletion in wild type and excision repair-deficient Escherichia coli strains (using a genetic assay for deletions). The rate of triplet repeat deletion decreased in an E. coli strain deficient in the damage recognition protein UvrA. Moreover, loops containing 23 CTG repeats were less efficiently excised from heteroduplex plasmids after their transformation into the uvrA(-) strain. As a result, an increased proportion of plasmids containing the full-length repeat were recovered after the replication of heteroduplex plasmids containing unrepaired loops. In biochemical experiments, UvrA bound to heteroduplex substrates containing repeat loops of 1, 2, or 17 CAG repeats with a K(d) of about 10-20 nm, which is an affinity about 2 orders of magnitude higher than that of UvrA bound to the control substrates containing (CTG)(n).(CAG)(n) in the linear form. These results suggest that UvrA is involved in triplet repeat instability in cells. Specifically, UvrA may bind to loops formed during replication slippage or in slipped strand DNA and initiate DNA repair events that result in repeat deletion. These results imply a more comprehensive role for UvrA, in addition to the recognition of DNA damage, in maintaining the integrity of the genome.     

Variable germline and embryonic instability of the human minisatellite MS32 (D1S8) in transgenic mice.

Tandem repeat loci such as minisatellites and trinucleotide repeats frequently show instability. We have investigated mutation at human minisatellite MS32 (locus D1S8) transferred to transgenic mice. Three lines of hemizygous transgenic mice were studied. A single-copy line (110D) was seen to be relatively stable, whilst two multicopy lines showed structural instability of the transgene in pedigrees (lines 109 and 110A). For both these lines, mutant structures were detected as a result of mutation events having occurred in the germline or early embryo. Structural changes seen included gain or loss of minisatellite repeat units (110A and 109), alteration of DNA flanking the minisatellite repeat array (109 only) or deletion of the entire transgene (109 only). This work demonstrates that tandem repeat transgenes can show instability and thus provide additional systems for the analysis of repetitive DNA structural change in mice.     

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.     

Nuclease-deficient FEN-1 blocks Rad51/BRCA1-mediated repair and causes trinucleotide repeat instability

Previous studies have shown that expansion-prone repeats form structures that inhibit human flap endonuclease (FEN-1). We report here that faulty processing by FEN-1 initiates repeat instability in mammalian cells. Disease-length CAG tracts in Huntington's disease mice heterozygous for FEN-1 display a tendency toward expansions over contractions during intergenerational inheritance compared to those in homozygous wild-type mice. Further, with regard to human cells expressing a nuclease-defective FEN-1, we provide direct evidence that an unprocessed FEN-1 substrate is a precursor to instability. In cells with no endogenous defects in DNA repair, exogenous nuclease-defective FEN-1 causes repeat instability and aberrant DNA repair. Inefficient flap processing blocks the formation of Rad51/BRCA1 complexes but invokes repair by other pathways.     

CTG repeat instability and size variation timing in DNA repair-deficient mice

Type 1 myotonic dystrophy is caused by the expansion of an unstable CTG repeat in the DMPK gene. We have investigated the molecular mechanisms underlying the CTG repeat instability by crossing transgenic mice carrying >300 unstable CTG repeats in their human chromatin environment with mice knockout for genes involved in various DNA repair pathways: Msh2 (mismatch repair), Rad52 and Rad54 (homologous recombination) and DNA-PKcs (non-homologous end-joining). Genes of the non-homologous end-joining and homologous recombination pathways did not seem to affect repeat instability. Only lack of Rad52 led to a slight decrease in expansion range. Unexpectedly, the absence of Msh2 did not result in stabilization of the CTG repeats in our model. Instead, it shifted the instability towards contractions rather than expansions, both in tissues and through generations. Furthermore, we carefully analyzed repeat transmissions with different Msh2 genotypes to determine the timing of intergenerational instability. We found that instability over generations depends not only on parental germinal instability, but also on a second event taking place after fertilization.     

Mutation rates in the complex microsatellite MYCL1 and related simple repeats in cultured human cells

Microsatellite instability is a phenotype observed in tumors cells that have defects in DNA mismatch repair (MMR). Most markers used for detecting microsatellite instability are mono- and dinucleotide repeats, but one tetranucleotide repeat (MYCL1) has been reported to be useful for this purpose. The MYCL1 repeat is actually a complex repeat, made up of approximately 14 GAAA tetranucleotides plus various other GA-rich repeats. In order to determine the nature of the instability of the this sequence, we have used a frameshift-reversion assay in MMR-proficient and -deficient human cells to compare the mutation rates and the types of mutation of MYCL1 to those of the related simple repeats (GAAA)17, (GA)17, and (CA)17. We found that the complex repeat was the most stable of the repeats examined in cells deficient in MMR; the tetranucleotide was less stable, while the dinucleotides were the least stable. In MMR-proficient cells, the relative rates were reversed; the MYCL1 repeat was the least stable, the tetranucleotide was more stable, and the dinucleotides were the most stable. These results suggest that MYCL1 and the pure tetranucleotide have relatively low rates of errors during replication, but that the errors in these repeats are corrected less efficiently than those in the smaller repeats. Because of their high rate of instability in MMR-proficient cells, MYCL1 and other tetranucleotide repeats appear to lack specificity for detection of tumors with defective MMR.