Effects of temperature, Mg2+ concentration and mismatches on triplet-repeat expansion during DNA replication in vitro

Lyons-Darden, Tara; Topal, Michael D.

doi:10.1093/nar/27.11.2235

Cited by 25 publications

(24 citation statements)

References 41 publications

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“…The former mimics the sliding model (6, 7) for expansion by DNA slippage and the latter mimics models involving bulge and hairpin intermediates (14 -18). Replication from a floating primer generates long TREs under conditions of low Mg 2ϩ (29). The presence of unique sequences flanking the repeat tract, however, significantly reduces the amount of TRE.…”

Section: Resultsmentioning

confidence: 99%

Abasic Sites Induce Triplet-repeat Expansion during DNA Replication in Vitro

Lyons-Darden

Topal

1999

Journal of Biological Chemistry

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The occurrence of triplet-repeat expansion (TRE) during transmission of genetic information is involved in many neurological and neuromuscular diseases including Fragile X syndrome and myotonic dystrophy. DNA slippage during replicative synthesis appears to cause TRE. The causes of DNA slippage, however, remain mostly unknown. We investigated the effects of abasic sites on the occurrence of TRE during DNA replication in vitro using Escherichia coli Klenow polymerase I as the model polymerase. Here we show that a single abasic site analog, synthesized in the triplet-repeat tract at the 5 end of the template strand, induced dramatic TRE during DNA synthesis. The amount of TRE induced decreased when the abasic site was moved to the middle of the repeat tract, consistent with effectively decreasing the length of the repeat tract. Placing the abasic site in the primer did not induce TRE. TRE was sequence-dependent. The damage-induced increase in growing strand TRE depended on the sequence of the growing strand repeat as AAT ϳ ATT > CAG > CTG. The expansions required replication from a primer complementary to the repeat tract. The expanded tracts were sequenced and contained multiple additions of the original repeat. The results imply that DNA damage can play a significant role in generating TRE in vivo.The occurrence of triplet-repeat expansion (TRE) 1 during transmission of genetic information is involved in many diseases including Fragile X syndrome (1-3), the most common form of mental retardation; myotonic dystrophy, a neuromuscular disorder; and several neurodegenerative disorders (1, 2). DNA slippage (4) during replicative synthesis is believed to be a major contributor to TRE in vivo (5-8), because elimination of recombination in yeast (9) and in Escherichia coli (10) does not influence TRE, whereas elimination of mismatch repair does (10, 11). Pausing or blockage of DNA replication has been proposed to promote slippage by giving the DNA replication complex more time to dissociate and form misaligned DNA intermediates (12, 13). Hairpins (14 -18), bulges (19,20), tetraplexes (18, 21), and possibly "slipped" structures (22, 23) have been proposed to promote DNA slippage. The structures were proposed to act either as intermediates, by their formation within the repeat or by blocking or pausing replication toward the end of the repeat (12, 24) or a combination of both (13,17,24). Replication pause sites are hot spots for nucleotide misincorporation (25). If pausing also induces slippage, the occurrence within the repeat tract of DNA damage that blocks DNA replication could profoundly effect TRE.Studies of replication of DNA template-primers in vitro with DNA polymerase alone are important for their potential ability to uncover mutation mechanisms. Replication of triplet-repeat tracts in vitro shows TRE (26 -29). Here, we report the effects of tract length, tract sequence, and DNA damage on TRE during DNA replication in vitro. The abasic site analog tetrahydrofuran (THF) was used as the model DNA damage lesion. A...

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Section: Resultsmentioning

confidence: 99%

Abasic Sites Induce Triplet-repeat Expansion during DNA Replication in Vitro

Lyons-Darden

Topal

1999

Journal of Biological Chemistry

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“…For example, interruptions in the CTG/CAG and CCG/CGG tracts found in the SCA1 and FRAXA loci suppress expansion potential in humans, and when expansions are observed they are usually accompanied by loss of the interruptions and generation of a long pure tract (reviewed in . Interruptions destabilize hairpins, slipped strand structures and CGG quadruplexes in vitro (Gacy et al, 1995;Pearson et al, 1998a;Lyons-Darden and Topal, 1999b;Weisman-Shomer et al, 2000a). Interruptions also appear to decrease the frequency of tract length changes in vivo in model organisms Shimizu et al, 1996;Hirst and White, 1998;Maurer et al, 1998;Jakupciak and Wells, 1999;White et al, 1999), and a systematic study into the influence of interruptions on CTG/ CAG expansion rates found that an ATGATG interruption in a (CTG) 25 tract can stabilize the tract by as much as 90 fold .…”

Section: Length Purity and Chromosomal Contextmentioning

confidence: 99%

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

Lenzmeier

Freudenreich

2003

Cytogenet Genome Res

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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 double-stranded 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 contractions 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.

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“…Thus, expanded TTC repeat means that the TTC growing strand grew beyond the length expected from replication of the template strand.) The DNA sequences of the type I GCC/GGC expansions (35) and the type II expansions by Klenow pol I (29,38) were sequenced to provide evidence that the expanded reaction products completely mimic the growing strand TR sequence. Such demonstrations are important because other possible DNA expansion pathways, not based on DNA slippage, are predicted to give products during DNA replication in vitro that vary from the growing strand TR sequence (22,43).…”

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confidence: 99%

Hairpin Formation in Friedreich's Ataxia Triplet Repeat Expansion

Heidenfelder¹,

Makhov²,

Topal³

2003

Journal of Biological Chemistry

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Triplet repeat tracts occur throughout the human genome. Expansions of a (GAA) n /(TTC) n repeat tract during its transmission from parent to child are tightly associated with the occurrence of Friedreich's ataxia. Evidence supports DNA slippage during DNA replication as the cause of the expansions. DNA slippage results in single-stranded expansion intermediates. Evidence has accumulated that predicts that hairpin structures protect from DNA repair the expansion intermediates of all of the disease-associated repeats except for those of Friedreich's ataxia. How the latter repeat expansions avoid repair remains a mystery because (GAA) n and (TTC) n repeats are reported not to self-anneal. To characterize the Friedreich's ataxia intermediates, we generated massive expansions of (GAA) n and (TTC) n during DNA replication in vitro using human polymerase ␤ and the Klenow fragment of Escherichia coli polymerase I. Electron microscopy, endonuclease cleavage, and DNA sequencing of the expansion products demonstrate, for the first time, the occurrence of large and growing (GAA) n and (TTC) n hairpins during DNA synthesis. The results provide unifying evidence that predicts that hairpin formation during DNA synthesis mediates all of the disease-associated, triplet repeat expansions.

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Effects of temperature, Mg2+ concentration and mismatches on triplet-repeat expansion during DNA replication in vitro

Cited by 25 publications

References 41 publications

Abasic Sites Induce Triplet-repeat Expansion during DNA Replication in Vitro

Abasic Sites Induce Triplet-repeat Expansion during DNA Replication in Vitro

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

Hairpin Formation in Friedreich's Ataxia Triplet Repeat Expansion

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