Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells (original) (raw)

Replication-dependent instability at (CTG)•(CAG) repeat hairpins in human cells

Nature Chemical Biology, 2010

Instability of (CTG)•(CAG) microsatellite trinucleotide repeat (TNR) sequences is responsible for more than one dozen neurological or neuromuscular diseases. TNR instability during DNA synthesis is thought to involve slipped strand or hairpin structures in template or nascent DNA strands, although direct evidence for hairpin formation in human cells is lacking. We have used targeted recombination to create a series of isogenic HeLa cell lines in which (CTG)•(CAG) repeats are replicated from an ectopic copy of the c-myc replication origin. In this system the tendency of chromosomal (CTG)•(CAG) tracts to expand or contract was affected by origin location and the leading or lagging strand replication orientation of the repeats, and instability was enhanced by prolonged cell culture, increasing TNR length, and replication inhibition. Hairpin cleavage by synthetic zinc finger nucleases in these cells has provided the first direct evidence for the formation of hairpin structures during replication in vivo.

Features of trinucleotide repeat instability in vivo

Cell Research, 2008

Unstable repeats are associated with various types of cancer and have been implicated in more than 40 neurodegenerative disorders. Trinucleotide repeats are located in non-coding and coding regions of the genome. Studies of bacteria, yeast, mice and man have helped to unravel some features of the mechanism of trinucleotide expansion. Looped DNA structures comprising trinucleotide repeats are processed during replication and/or repair to generate deletions or expansions. Most in vivo data are consistent with a model in which expansion and deletion occur by different mechanisms. In mammals, microsatellite instability is complex and appears to be influenced by genetic, epigenetic and developmental factors.

Mechanism of Trinucleotide Repeats Instabilities: The Necessities

The mechanism underlying CAGCTG CGGCCG and GAA.TTC trinucleotide repeats expansion and contraction instabilities has not been clearly understood. Investigations in vitro have demonstrated that the disease causing repeats are capable of adopting non-B secondary structures that mediate repeats expansion. However, in vivo, similar observations have not been easily made so far. Investigations on the non-B secondary structure formation using E.coli, yeast etc cannot simulate the suggested repeats expansion instability. These could leave a space to infer a disassociation of the suggested repeats non-B secondary structure formation and the repeats expansion in vivo. Although longer trinucleotide repeats may be theoretically easier to form non-B DNA secondary structures in replication or in post-replication, however such non-B secondary structures are likely to cause repeat fragility rather than repeat expansion. In fact, repeat expansion as seen in patients may not necessarily require trinucleotide repeats to form non-B secondary structures, instead the repeat expansions can be produced through a RNA transcription-stimulated local repeat DNA replication and a subsequent DNA rearrangement.

Trinucleotide repeat instability: a hairpin curve at the crossroads of replication, recombination, and repair

Cytogenetic and Genome Research, 2003

The trinucleotide repeats that expand to cause human disease form hairpin structures in vitro that are proposed to be the major source of their genetic instability in vivo. If a replication fork is a train speeding along a track of doublestranded DNA, the trinucleotide repeats are a hairpin curve in the track. Experiments have demonstrated that the train can become derailed at the hairpin curve, resulting in significant damage to the track. Repair of the track often results in contrac-tions and expansions of track length. In this review we introduce the in vitro evidence for why CTG/CAG and CCG/CGG repeats are inherently unstable and discuss how experiments in model organisms have implicated the replication, recombination and repair machinery as contributors to trinucleotide repeat instability in vivo.

Instability of (CTG)n•(CAG)n trinucleotide repeats and DNA synthesis

Cell & bioscience, 2012

Expansion of (CTG)n•(CAG)n trinucleotide repeat (TNR) microsatellite sequences is the cause of more than a dozen human neurodegenerative diseases. (CTG)n and (CAG)n repeats form imperfectly base paired hairpins that tend to expand in vivo in a length-dependent manner. Yeast, mouse and human models confirm that (CTG)n•(CAG)n instability increases with repeat number, and implicate both DNA replication and DNA damage response mechanisms in (CTG)n•(CAG)n TNR expansion and contraction. Mutation and knockdown models that abrogate the expression of individual genes might also mask more subtle, cumulative effects of multiple additional pathways on (CTG)n•(CAG)n instability in whole animals. The identification of second site genetic modifiers may help to explain the variability of (CTG)n•(CAG)n TNR instability patterns between tissues and individuals, and offer opportunities for prognosis and treatment.

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

PLoS ONE, 2013

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 59-end of CTG repeats resulted in expansion, whereas a lesion located either in the middle or the 39-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 b 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.

The complex pathology of trinucleotide repeats

Current Opinion in Cell Biology, 1997

The expansion of trinucleotide repeat sequences has now been shown to be the underlying cause of at least ten human disorders. Unifying features among these diseases include the unstable behavior of the triplet repeat during germline transmission when the length of the repeat exceeds a critical value. However, the trinucleotide repeat disorders can be divided into two distinct groups. Type I disorders involve the expansion of CAG repeats, which encode an expanded polyglutamine, inserted into the open-reading frame of a gene that is usually quite broadly expressed. Recently, mouse models for type I disorders have been developed and the basis of pathology is under study, both in these models and through biochemical and cell biological approaches. The type II disorders involve repeat expansions in noncoding regions of genes. The mechanisms by which these repeat expansions lead to pathology may be quite diverse.

Mutations in Yeast Replication Proteins That Increase CAG/CTG Expansions Also Increase Repeat Fragility

Molecular and Cellular Biology, 2003

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 laggingstrand replication: Fen1/Rad27, an endo/exonuclease involved in Okazaki fragment maturation, the nuclease/ helicase Dna2, RNase HI, DNA ligase, polymerase ␦, 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.

Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair

DNA Repair, 2016

-CAG/CTG triplet repeats block replication in both orientations on a yeast chromosome -Replication fork stalling depends on mismatch repair integrity -Msh2p is enriched at CAG/CTG triplet repeats in both orientations -MSH2 overexpression favors or stabilizes the formation of heteroduplex molecules Abstract Trinucleotide repeat expansions are responsible for at least two dozen neurological disorders.

Instability of repetitive DNA sequences: the role of replication in multiple mechanisms

… Academy of Sciences of the United …, 2001

Rearrangements between tandem sequence homologies of various lengths are a major source of genomic change and can be deleterious to the organism. These rearrangements can result in either deletion or duplication of genetic material flanked by direct sequence repeats. Molecular genetic analysis of repetitive sequence instability in Escherichia coli has provided several clues to the underlying mechanisms of these rearrangements. We present evidence for three mechanisms of RecA-independent sequence rearrangements: simple replication slippage, sister-chromosome exchange-associated slippage, and single-strand annealing. We discuss the constraints of these mechanisms and contrast their properties with RecA-dependent homologous recombination. Replication plays a critical role in the two slipped misalignment mechanisms, and difficulties in replication appear to trigger rearrangements via all these mechanisms.