Christopher E. Pearson

TRINUCLEOTIDE REPEAT DNA STRUCTURES : DYNAMIC MUTATIONS FROM DYNAMIC DNA

Models for the disease-associated expansion of (CTG)n.(CAG)n, (CGG)n.(CCG)n, and (GAA)n.(TTC)n trinucleotide repeats involve alternative DNA structures formed during DNA replication, repair and recombination. These repeat sequences are inherently flexible and can form a variety of hairpins, intramolecular triplexes, quadruplexes, and slipped-strand structures that may be important intermediates and result in their genetic instability.

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 β 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.

Isolated short CTG/CAG DNA slip-outs are repaired efficiently by hMutSβ, but clustered slip-outs are poorly repaired

Expansions of CTG/CAG trinucleotide repeats, thought to involve slipped DNAs at the repeats, cause numerous diseases including myotonic dystrophy and Huntingtons disease. By unknown mechanisms, further repeat expansions in transgenic mice carrying expanded CTG/CAG tracts require the mismatch repair (MMR) proteins MSH2 and MSH3, forming the MutSβ complex. Using an in vitro repair assay, we investigated the effect of slip-out size, with lengths of 1, 3, or 20 excess CTG repeats, as well as the effect of the number of slip-outs per molecule, on the requirement for human MMR. Long slip-outs escaped repair, whereas short slip-outs were repaired efficiently, much greater than a G-T mismatch, but required hMutSβ. Higher or lower levels of hMutSβ or its complete absence were detrimental to proper repair of short slip-outs. Surprisingly, clusters of as many as 62 short slip-outs (one to three repeat units each) along a single DNA molecule with (CTG)50•(CAG)50 repeats were refractory to repair, and repair efficiency was reduced further without MMR. Consistent with the MutSβ requirement for instability, hMutSβ is required to process isolated short slip-outs; however, multiple adjacent short slip-outs block each others repair, possibly acting as roadblocks to progression of repair and allowing error-prone repair. Results suggest that expansions can arise by escaped repair of long slip-outs, tandem short slip-outs, or isolated short slip-outs; the latter two types are sensitive to hMutSβ. Poor repair of clustered DNA lesions has previously been associated only with ionizing radiation damage. Our results extend this interference in repair to neurodegenerative disease-causing mutations in which clustered slip-outs escape proper repair and lead to expansions.

Unexpected formation of parallel duplex in GAA and TTC trinucleotide repeats of Friedreich’s ataxia

The onset and progress of Friedreichs ataxia (FRDA) is associated with the genetic instability of the (GAA).(TTC) trinucleotide repeats located within the frataxin gene. The instability of these repeats may involve the formation of an alternative DNA structure. Poly-purine (R)/poly-pyrimidine (Y) sequences typically form triplex DNA structures which may contribute to genetic instability. Conventional wisdom suggested that triplex structures formed by these poly-purine (R)/poly-pyrimidine (Y) sequences may contribute to their genetic instability. Here, we report the characterization of the single-stranded GAA and TTC sequences and their mixtures using NMR, UV-melting, and gel electrophoresis, as well as chemical and enzymatic probing methods. We show that the FRDA GAA/TTC, repeats are capable of forming various alternative structures. The most intriguing is the observation of a parallel (GAA).(TTC) duplex in equilibrium with the antiparallel Watson-Crick (GAA).(TTC) duplex. We also show that the GAA strands form self-assembled structures, whereas the TTC strands are essentially unstructured. Finally, we demonstrate that the FRDA repeats form only the YRY triplex (but not the RRY triplex) at neutral pH and the complete formation of the YRY triplex requires the ratio of GAA to TTC strand larger than 1:2. The structural features presented here and in other studies distinguish the FRDA (GAA)¿(TTC) repeats from the fragile X (CGG).CCG), myotonic dystrophy (CTG).(CAG) and the Huntington (CAG).(CTG) repeats.

CTG/CAG Repeat Instability Is Modulated by the Levels of Human DNA Ligase I and Its Interaction with Proliferating Cell Nuclear Antigen: A DISTINCTION BETWEEN REPLICATION AND SLIPPED-DNA REPAIR*

Mechanisms contributing to disease-associated trinucleotide repeat instability are poorly understood. DNA ligation is an essential step common to replication and repair, both potential sources of repeat instability. Using derivatives of DNA ligase I (hLigI)-deficient human cells (46BR.1G1), we assessed the effect of hLigI activity, overexpression, and its interaction with proliferating cell nuclear antigen (PCNA) upon the ability to replicate and repair trinucleotide repeats. Compared with LigI+/+, replication progression through repeats was poor, and repair tracts were broadened beyond the slipped-repeat for all mutant extracts. Increased repeat instability was linked only to hLigI overexpression and expression of a mutant hLigI incapable of interacting with PCNA. The endogenous mutant version of hLigI with reduced ligation activity did not alter instability. We distinguished the DNA processes through which hLigI contributes to trinucleotide instability. The highest levels of repeat instability were observed under the hLigI overexpression and were linked to reduced slipped-DNAs repair efficiencies. Therefore, the replication-mediated instability can partly be attributed to errors during replication but also to the poor repair of slipped-DNAs formed during this process. However, repair efficiencies were unaffected by expression of a PCNA interaction mutant of hLigI, limiting this instability to the replication process. The addition of purified proteins suggests that disruption of LigI and PCNA interactions influences trinucleotide repeat instability. The variable levels of age- and tissue-specific trinucleotide repeat instability observed in myotonic dystrophy patients and transgenic mice may be influenced by varying steady state levels of DNA ligase I in these tissues and during different developmental windows.

Interconverting Conformations of Slipped-DNA Junctions Formed by Trinucleotide Repeats Affect Repair Outcome

Expansions of (CTG)·(CAG) repeated DNAs are the mutagenic cause of 14 neurological diseases, likely arising through the formation and processing of slipped-strand DNAs. These transient intermediates of repeat length mutations are formed by out-of-register mispairing of repeat units on complementary strands. The three-way slipped-DNA junction, at which the excess repeats slip out from the duplex, is a poorly understood feature common to these mutagenic intermediates. Here, we reveal that slipped junctions can assume a surprising number of interconverting conformations where the strand opposite the slip-out either is fully base paired or has one or two unpaired nucleotides. These unpaired nucleotides can also arise opposite either of the nonslipped junction arms. Junction conformation can affect binding by various structure-specific DNA repair proteins and can also alter correct nick-directed repair levels. Junctions that have the potential to contain unpaired nucleotides are repaired with a significantly higher efficiency than constrained fully paired junctions. Surprisingly, certain junction conformations are aberrantly repaired to expansion mutations: misdirection of repair to the non-nicked strand opposite the slip-out leads to integration of the excess slipped-out repeats rather than their excision. Thus, slipped-junction structure can determine whether repair attempts lead to correction or expansion mutations.

Table S2: Trans-factors and trinucleotide repeat instability Trans-factor

The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation as a foundation for investigating the relationship between genotype and phenotype. Here we present results of the pilot phase of the project, designed to develop and compare different strategies for genome-wide sequencing with high-throughput platforms. We undertook three projects: low-coverage whole-genome sequencing of 179 individuals from four populations; high-coverage sequencing of two mother–father–child trios; and exon-targeted sequencing of 697 individuals from seven populations. We describe the location, allele frequency and local haplotype structure of approximately 15 million single nucleotide polymorphisms, 1 million short insertions and deletions, and 20,000 structural variants, most of which were previously undescribed. We show that, because we have catalogued the vast majority of common variation, over 95% of the currently accessible variants found in any individual are present in this data set. On average, each person is found to carry approximately 250 to 300 loss-of-function variants in annotated genes and 50 to 100 variants previously implicated in inherited disorders. We demonstrate how these results can be used to inform association and functional studies. From the two trios, we directly estimate the rate of de novo germline base substitution mutations to be approximately 10−8 per base pair per generation. We explore the data with regard to signatures of natural selection, and identify a marked reduction of genetic variation in the neighbourhood of genes, due to selection at linked sites. These methods and public data will support the next phase of human genetic research.

CHAPTER 41 – Error-Prone Repair of Slipped (CTG)·(CAG) Repeats and Disease-Associated Expansions

Disease-associated repeat instability can occur in various patient tissues, including germ cells, proliferating somatic tissues, and nonproliferating tissues like the brain. Various DNA metabolic processes could potentially give rise to slipped DNAs at the repeats—the supposed mutagenic intermediate of repeat instability. Transgenic mice with (CTG)·(CAG) repeats have revealed a requirement for several mismatch repair genes for the spontaneous expansion of the repeats. Results with an in vitro repair assay using human cell extracts and structurally defined slipped DNAs have revealed several distinct repair outcomes: correct repair, escaped repair, and error-prone repair, some of which can give rise to the expansion bias occurring in patients. These processes can be mediated by neuron-like cells supporting their contribution to instability in patient brains. Notably, the mismatch and nucleotide excision repair proteins such as hMSH2, hMSH3, hMLHl, XPF, or XPG were not involved in processing of the slip outs. This suggests that, if these proteins are involved in expansion, their role precedes the step of slip-out processing.

23 DNA Replication, Repeat Instability, and Human Disease

Alterations in the size of simple repetitive sequences, mostly CNG (where N = A, C, T, or G), within specific human genes comprise the disease-causing mutation for a growing class of inherited neurological, neurodegenerative, and neuromuscular disorders (Fig. 1). At least 36 human disorders are attributed to repeat instability, including Huntington’s disease (HD), myotonic dystrophy type 1 (DM1), and fragile X mental retardation syndrome (FRAXA) (see Appendix, Table VI). Depending on the disorder, repeat instability can be observed in somatic and germ-line tissues for both nonproliferative and proliferative cells, suggesting that a variety of DNA metabolic processes underlie the mutational process. Evidence indicates that DNA replication, independently and in conjunction with other metabolic processes, actively contributes to repeat instability. The contributions of DNA repair, features of instability in patients, and disease pathogenesis have been reviewed elsewhere (Cleary and Pearson 2003; Gatchel and Zoghbi 2005; Pearson et al. 2005). This chapter focuses on the contribution of DNA replication to repeat instability, primarily for trinucleotide repeats (TNRs). DYNAMIC MUTATION TNR instability is a “dynamic mutation” (Richards and Sutherland 1992) caused by the increased propensity of the expanded product of a mutation to undergo further expansion mutation. Within the general population, TNR tracts are typically short, polymorphic, and stably transmitted, only becoming unstable above a stability threshold length, usually 34–45 repeats. Transmission (and subsequent expansion) of the expanded repeat tract across multiple generations results in genetic anticipation, a decrease in age of onset, and increase in disease severity that is characteristic of...