Molecular Basis of Genetic Instability of Triplet Repeats (1996)

A veritable explosion is taking place in our understanding of the human genetics, biochemistry, and DNA structural issues related to human hereditary neuromuscular and neurodegenerative diseases. Also, the non-Mendelian expansion process that elicits these disease manifestations (anticipation) is under intense investigation. Within the last 3 years, the molecular basis of 10 human genetic disorders (including fragile X syndrome (FRAXA and FRAXE), myotonic dystrophy (DM), ( 1The abbreviations used are: DMmyotonic dystrophyHDHuntington's diseaseSCA1spinocerebellar ataxia type 1DRPLAdentatorubral-pallidoluysian atrophybpbase pair(s)SBMAspinobulbar muscular atrophy.) Kennedy's disease, Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), and dentatorubral-pallidoluysian atrophy (DRPLA)) has been partially established (reviewed in (1.Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar, 2.Bates G. Lehrach H. BioEssays. 1994; 16: 277-284Crossref PubMed Scopus (112) Google Scholar, 3.Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar, 4.Panzer S. Kuhl D.P.A. Caskey C.T. Stem Cells. 1995; 13: 146-157Crossref PubMed Scopus (27) Google Scholar, 5.Krahe R. Ashizawa T. Wetherall J. Groth D. Hypervariable Genetic Markers. CRC Press, Inc., Boca Raton, FL1995: 29-60Google Scholar)). The diseases are characterized at the molecular level by the expansion of a simple triplet repeat (CTG and CGG) from less than 15 copies of the repeat in normal individuals to scores of copies in affected cases; thousands of copies are found in some cases of fragile X and myotonic dystrophy. These increases in size occur upon passage of an expanded repeat in the chromosome to offspring. Moreover, the symptoms of these diseases follow an unusual genetic pattern called anticipation, in which the disease becomes more severe and has an earlier age of onset with each successive generation (reviewed in (1.Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar, 2.Bates G. Lehrach H. BioEssays. 1994; 16: 277-284Crossref PubMed Scopus (112) Google Scholar, 3.Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar, 4.Panzer S. Kuhl D.P.A. Caskey C.T. Stem Cells. 1995; 13: 146-157Crossref PubMed Scopus (27) Google Scholar, 5.Krahe R. Ashizawa T. Wetherall J. Groth D. Hypervariable Genetic Markers. CRC Press, Inc., Boca Raton, FL1995: 29-60Google Scholar, 6.Wells R.D. Sinden R.R. Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 107-138Google Scholar)). The instability of repeats in the genome has also been linked to hereditary nonpolyposis colon cancer, which may involve mutations in mismatch repair functions(4.Panzer S. Kuhl D.P.A. Caskey C.T. Stem Cells. 1995; 13: 146-157Crossref PubMed Scopus (27) Google Scholar, 5.Krahe R. Ashizawa T. Wetherall J. Groth D. Hypervariable Genetic Markers. CRC Press, Inc., Boca Raton, FL1995: 29-60Google Scholar, 7.Thibodeau S.N. Bren G. Schaid D. Science. 1993; 260: 816-819Crossref PubMed Scopus (2812) Google Scholar, 8.Shibata D. Peinado M.A. Ionov Y. Malkhosyan S. Perucho M. Nat. Genet. 1994; 6: 273-281Crossref PubMed Scopus (455) Google Scholar, 9.Baker S.M. Bronner C.E. Zhang L. Plug A.W. Robatzek M. Warren G. Elliott E.A. Yu J. Ashley T. Arnheim N. Flavell R.A. Liskay R.M. Cell. 1995; 82: 309-319Abstract Full Text PDF PubMed Scopus (476) Google Scholar). myotonic dystrophy Huntington's disease spinocerebellar ataxia type 1 dentatorubral-pallidoluysian atrophy base pair(s) spinobulbar muscular atrophy. For example, Huntington's disease shows anticipation and has expanded CAG triplet repeats(1.Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar, 2.Bates G. Lehrach H. BioEssays. 1994; 16: 277-284Crossref PubMed Scopus (112) Google Scholar, 3.Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar, 4.Panzer S. Kuhl D.P.A. Caskey C.T. Stem Cells. 1995; 13: 146-157Crossref PubMed Scopus (27) Google Scholar, 5.Krahe R. Ashizawa T. Wetherall J. Groth D. Hypervariable Genetic Markers. CRC Press, Inc., Boca Raton, FL1995: 29-60Google Scholar, 10.Huntington's Disease Collaborative Research GroupCell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (7118) Google Scholar). A CAG repeat of between 11 and 34 bp in the normal population encodes a polyglutamine tract in the IT15 gene. Expansion to about 90 bp occurs in HD patients. The age of onset correlates with the length of the triplet repeat with the largest changes in repeat lengths seen upon paternal transmission(11.Duyao M. Ambrose C. Myers R. Novelletto A. Persichetti F. Frontali M. Folstein S. Ross C. Franz M. Abbott M. Gray J. Conneally P. Young A. Penney J. Hollingsworth Z. Shoulson I. Lazzarini A. Falek A. Koroshetz W. Sax D. Bird E. Vonsattel J. Bonilla E. Alvir J. Conde J.Bickham Cha J.-H. Dure L. Gomex F. Ramos M. Sanchez-Ramos J. Snodgrass S. de Young M. Wexler N. Moscowitz C. Penchaszadeh G. MacFarlane H. Anderson M. Jenkins B. Srinidhi J. Barnes G. Gusella J. MacDonald M. Nat. Genet. 1993; 4: 387-392Crossref PubMed Scopus (904) Google Scholar). Sperm display a heterogeneous expanded repeat length. An intermediate allele, IA, containing 30-38 (or 34-38) repeats (perhaps similar to a premutation in fragile X or DM) has been identified. Initial reports suggest that sporadic expansion of the IA allele occurs only through paternal transmission(12.Goldberg Y.P. Kremer B. Andrew S.E. Theilmann J. Graham R.K. Squitieri F. Telenius H. Adam S. Sajoo A. Starr E. Heiberg A. Wolff G. Hayden M.R. Nat. Genet. 1993; 5: 174-179Crossref PubMed Scopus (235) Google Scholar, 13.Myers R.H. MacDonald M.E. Koroshetz W.J. Duyao M.P. Ambrose C.M. Taylor S.A.M. Barnes G. Srinidhi J. Lin C.S. Whaley W.L. Lazzarini A.M. Schwarz M. Wolff G. Bird E.D. Vonsattel J.-P. G. Gusella J.F. Nat. Genet. 1993; 5: 168-173Crossref PubMed Scopus (227) Google Scholar). The function of the gene product is uncertain(14.Trottier Y. Devys D. Imbert G. Sandou F. An I. Lutz Y. Weber C. Agid Y. Hirsch E.C. Mandel J.-L. Nat. Genet. 1995; 10: 104-110Crossref PubMed Scopus (382) Google Scholar). Considering Mendelian genetic principles, anticipation was an enigma. The discovery of expanding triplet repeats (or "mutable mutations") in diseases showing anticipation afforded a physical basis for this unusual genetic phenomenon. Expansion of the triplet repeat is responsible for the genetic defect, influencing the activity of a glutamine-containing protein (SBMA, HD, SCA1, and DRPLA) or influencing the level of expression of a gene with which the repeat is associated (fragile X and DM)(1.Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar, 2.Bates G. Lehrach H. BioEssays. 1994; 16: 277-284Crossref PubMed Scopus (112) Google Scholar, 3.Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar, 4.Panzer S. Kuhl D.P.A. Caskey C.T. Stem Cells. 1995; 13: 146-157Crossref PubMed Scopus (27) Google Scholar, 5.Krahe R. Ashizawa T. Wetherall J. Groth D. Hypervariable Genetic Markers. CRC Press, Inc., Boca Raton, FL1995: 29-60Google Scholar). All triplet repeat genetic diseases identified to date show anticipation. Several other diseases also show anticipation including spinocerebellar ataxia type 2(15.Pulst S.-M. Nechiporuk A. Starkman S. Nat. Genet. 1993; 5: 8-10Crossref PubMed Scopus (65) Google Scholar), bipolar affective disorder(16.McInnis M.G. McMahon F.J. Chase G.A. Simpson S.G. Ross C.A. DePaulo Jr., J.R. Am. J. Hum. Genet. 1993; 53: 385-390PubMed Google Scholar), and hereditary spastic paraparesis (Strumpell's disease)(17.Bruyn R.P.M. van Deutekom J. Frants R.R. Padberg G.W. Clin. Neurol. Neurosurg. 1993; 95: 125-129Crossref PubMed Scopus (22) Google Scholar). If a correlation exists between anticipation and triplet repeats, many more diseases showing anticipation may be identified since there are more than 40 genes containing associated triplet repeats. An understanding of the molecular mechanisms of triplet repeat instabilities (expansions and deletions) is important for the comprehension of anticipation. Kang et al.(18.Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar) have established a defined genetic system that shows promise for the dissection of this process. The frequency of genetic expansions or deletions in Escherichia coli depends on the direction of replication(18.Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar). Large expansions occur predominantly when the CTGs are in the leading template strand rather than the lagging strand. However, deletions are more prominent when the CTGs are in the opposite orientation (Fig. 1). Most deletions generate products of defined size classes. Strand slippage coupled with non-classical DNA structures (Fig. 2) probably accounts for these observations and relates to expansion-deletion mechanisms in eukaryotic chromosomes. To study expansions, these workers determined if a plasmid that contains (CTG)130 is completely homogenous as a cloned molecule or if deletions and expansions had occurred that gave rise to sequence heterogeneity, even in a tiny percent of the molecules. The insert containing the triplet repeat was excised from the vector and separated by gel electrophoresis. The regions of the gel either above or below the insert band were eluted and "recloned"; recombinant plasmids were obtained that contained successively larger or smaller inserts, respectively. The family of inserts characterized by these methods contained repeat units ranging from 17 to 300. Hence, expansion and deletion occur in E. coli. This discovery lays the foundation for evaluating host cell genetic factors (replication, recombination, mismatch repair, etc.) that may elicit genetic instabilities. DNA sequence analyses showed that expansion and contraction always occurred in multiple repeats of 3 bp. Prior investigations (19.Jaworski A. Higgins N.P. Wells R.D. Zacharias W. J. Biol. Chem. 1991; 266: 2576-2581Abstract Full Text PDF PubMed Google Scholar) showed that deletions in dinucleotide repeat sequences occurred in multiple units of 2 bp.Figure 2:DNA structures of triplet repeats. Slipped structures and toroids are not mutually excluded.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fig. 1 outlines a possible mechanism for the expansion and deletion behaviors. For expansion, a hairpin loop may form on the lagging strand nascent DNA (CTG strand). NMR investigations (20.Smith G.K. Jie J. Fox G.E. Gao X. Nucleic Acids Res. 1995; 23: 4303-4311Crossref PubMed Scopus (66) Google Scholar) revealed that CTG oligomers form a stable anti-parallel duplex with TT pairs, whereas the complementary CAG strand forms a metastable conformation. When the CTG is the lagging strand template (orientation II), a loop may form on the lagging strand that will be bypassed during DNA synthesis to generate deletions. Multiple slippages (6.Wells R.D. Sinden R.R. Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 107-138Google Scholar) may be promoted by an "idling polymerase" caused by a strong block such as a DNA structure or the presence of proteins(21.Yano-Yanagisawa H. Li Y. Wang H. Kohwi Y. Nucleic Acids Res. 1995; 23: 2654-2660Crossref PubMed Scopus (18) Google Scholar), which causes continuous slippage (primer realignment) resulting in the expansion of larger sequences. Other workers (22.Jeffreys A.J. Tamaki K. MacLeod A. Monckton D.G. Neil D.L. Armour J.A.L. Nat. Genet. 1994; 6: 136-145Crossref PubMed Scopus (458) Google Scholar) favor gene conversion events to explain germline mutations at human minisatellites. Evolutionary studies (23.Eichler E.E. Kunst C.B. Lungenbeel K.A. Ryder O.A. Davison D. Warren S.T. Nelson D.L. Nat. Genet. 1995; 11: 301-308Crossref PubMed Scopus (43) Google Scholar) on the cryptic FMR1 CGG repeat suggest that replication slippage and unequal crossing over have been operative for >150 million years. Ohshima et al.(24.Ohshima K. Kang S. Wells R.D. J. Biol. Chem. 1996; 271: 1853-1856Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) have recently discovered that the CTG triplet repeat is the dominant genetic expansion product in E. coli. This extraordinary discovery was made possible by the successful cloning and characterization of all 10 repeating triplet sequences. ( 2K. Ohshima and R. D. Wells, unpublished work.) The relative capacity of the 10 repeating triplet sequences to be expanded in E. coli(18.Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar) was explored with a competition study. Surprisingly, the CTG triplet repeat was expanded at least nine times more frequently than any of the other nine triplets(24.Ohshima K. Kang S. Wells R.D. J. Biol. Chem. 1996; 271: 1853-1856Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Low levels of expansion were found also for CGG, GTG, and GTC. Thus, the structure of the CTG repeat and/or its utilization by the DNA synthetic systems in vivo must be quite different from the other triplets. The surprising discovery that CTG triplet repeats are the dominant expansion products in E. coli, as found (1.Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar, 2.Bates G. Lehrach H. BioEssays. 1994; 16: 277-284Crossref PubMed Scopus (112) Google Scholar, 3.Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar, 4.Panzer S. Kuhl D.P.A. Caskey C.T. Stem Cells. 1995; 13: 146-157Crossref PubMed Scopus (27) Google Scholar, 5.Krahe R. Ashizawa T. Wetherall J. Groth D. Hypervariable Genetic Markers. CRC Press, Inc., Boca Raton, FL1995: 29-60Google Scholar) in clinical samples from human hereditary diseases, suggests the importance of DNA structural properties(25.Wells R.D. J. Biol. Chem. 1988; 263: 1095-1098Abstract Full Text PDF PubMed Google Scholar). Other investigations have revealed that duplex CTG and CGG repeats have unorthodox properties including nucleosome assembly(26.Wang Y.-H. Amirhaeri S. Kang S. Wells R.D. Griffith J. Science. 1994; 265: 669-671Crossref PubMed Scopus (211) Google Scholar), their capacity to cause DNA polymerases to pause within the repeat sequences(27.Kang S. Ohshima K. Shimizu M. Amirhaeri S. Wells R.D. J. Biol. Chem. 1995; 270: 27014-27021Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), as well as conformational features as revealed by helical repeat and polyacrylamide gel migrations ( 3R. Gellibolian, M. Shimizu, S. Amirhaeri, S. Kang, K. Ohshima, J. E. Larson, Y.-H. Fu, C. T. Caskey, B. A. Oostra, and R. D. Wells, unpublished observations.) (discussed below). Further elucidation of the CTG repeat structural features along with the genetic factors responsible for expansion may explain why most (8 out of 10) (1.Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar, 2.Bates G. Lehrach H. BioEssays. 1994; 16: 277-284Crossref PubMed Scopus (112) Google Scholar, 3.Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar, 4.Panzer S. Kuhl D.P.A. Caskey C.T. Stem Cells. 1995; 13: 146-157Crossref PubMed Scopus (27) Google Scholar, 5.Krahe R. Ashizawa T. Wetherall J. Groth D. Hypervariable Genetic Markers. CRC Press, Inc., Boca Raton, FL1995: 29-60Google Scholar) triplet repeat hereditary disease genes contain CTG repeats. Although other triplet repeats are found in the human genome (29.Gastier J.M. Pulido J.C. Sunden S. Brody T. Buetow K.H. Murray J.C. Weber J.L. Hudson T.J. Sheffield V.C. Duyk G.M. Hum. Mol. Genet. 1995; 4: 1829-1836Crossref PubMed Scopus (80) Google Scholar), the lengths are shorter (generally <15 repeats) than found for these disease genes. Other work (30.Kang, S., Ohshima, K., Jaworski, A., Wells, R. D. (1996) J. Mol. Biol., in pressGoogle Scholar) has shown that the CTG triplet repeat is expanded in E. coli distal to the replication origin as a single large event of ∼120 bp. In summary, these investigations (18.Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar, 24.Ohshima K. Kang S. Wells R.D. J. Biol. Chem. 1996; 271: 1853-1856Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 30.Kang, S., Ohshima, K., Jaworski, A., Wells, R. D. (1996) J. Mol. Biol., in pressGoogle Scholar) establish a genetically defined system for studying the molecular mechanisms of this non-Mendelian process. A recent report of a transgenic mouse model for SBMA (31.Bingham P.M. Scott M.O. Wang S. McPhaul M.J. Wilson E.M. Garbern J.Y. Merry D.E. Fischbeck K.H. Nat. Genet. 1995; 9: 191-196Crossref PubMed Scopus (126) Google Scholar) found no change in length with transmission. Bacterial systems may provide useful mechanistic information until a genetically defined eukaryotic system can be established. In fact, a number of similarities exist between the behaviors observed in humans and this E. coli system (reviewed in (24.Ohshima K. Kang S. Wells R.D. J. Biol. Chem. 1996; 271: 1853-1856Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar)). As an accidental discovery as part of chemical probe analyses, the pausing of DNA synthesis in vitro at specific loci in double-stranded CTG and CGG triplet repeats was found(27.Kang S. Ohshima K. Shimizu M. Amirhaeri S. Wells R.D. J. Biol. Chem. 1995; 270: 27014-27021Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The DNA syntheses of CTG triplets ranging from 17 to 180 and CGG repeats from 9 to 160 repeats in length were studied in vitro. Primer extensions using the Klenow fragment of DNA polymerase I, the modified T7 DNA polymerase (Sequenase), or the human DNA polymerase β paused strongly at specific loci in the CTG repeats. The pausings were abolished by heating at 70°C. As the length of the triplet repeats in duplex DNA, but not in single-stranded DNA, was increased, the magnitude of pausings increased. CGG triplet repeats also showed similar, but not identical, patterns of pausings. These results indicate that appropriate lengths of the triplets adopt a non-B conformation(s) that blocks DNA polymerase progression; the resultant idling polymerase may catalyze slippages (Fig. 1) to give expanded sequences and, hence, provide the molecular basis for this non-Mendelian genetic process. Also, recent in vivo replication studies in E. coli( 4S. M. Mirkin, personal communication.) with plasmids containing the CGG repeat revealed length-dependent pause sites. Other studies (32.Usdin K. Woodford K.J. Nucleic Acids Res. 1995; 23: 4202-4209Crossref PubMed Scopus (228) Google Scholar) with single-stranded (CGG)20 as a template suggest a K+-dependent structure (tetraplex) that serves as a barrier to DNA synthesis in vitro. Mismatch repair-deficient E. coli(33.Modrich P. Science. 1995; 266: 1959-1960Crossref Scopus Google Scholar) were studied in to the factors in genetic instabilities as well as DNA structural issues in CTG repeats are in plasmids in E. coli containing mutations in the mismatch repair genes or A. R. Kang S. Shimizu M. Sinden R.R. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). When plasmids containing were for about in or of the plasmids contained a whereas in the only about of the plasmids contained the The deletions occur only in the not in DNA the many products of the deletions are heterogeneous in deletion products of about and repeats were The E. coli mismatch repair during replication and generate single-stranded stable hairpin structures may which can be bypassed by DNA polymerase during the of duplex studies were with plasmids containing CGG no of these triplets was found in the mismatch repair and human mismatch repair are similar P. 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DNA and Press, Scholar)). For example, repeat sequences form and DNA, sequences adopt repeats form and repeating A exist in unusual structures were to exist in vivo in plasmids (6.Wells R.D. Sinden R.R. Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 107-138Google Scholar, R.R. DNA and Press, Scholar) and in S. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). Several recent studies were M. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, M. Yu A. J. 1995; 34 PubMed Scopus (112) Google Scholar, X. P. R. R.K. A. E.M. G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus (228) Google Scholar, A.M. G. N. S. C.T. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, Nucleic Acids Res. 1995; 23: PubMed Scopus Google Scholar, X. X. G.K. M. H. J. Am. 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Kang S. Wells R.D. Griffith J. Science. 1994; 265: 669-671Crossref PubMed Scopus (211) Google Scholar) since the (Fig. 2) provide a are the structural of and of bp of DNA about an of that containing lengths of CTG from to repeats were Y.-H. Amirhaeri S. Kang S. Wells R.D. Griffith J. Science. 1994; 265: 669-671Crossref PubMed Scopus (211) Google Scholar). The of nucleosome with expanded triplet blocks that such blocks may through the of stable (Fig. In fact, the expanded CTG triplet repeats are the nucleosome Y.-H. Griffith J. 1994; Scopus Google Scholar), even to the of the nucleosome sequences. In summary, that of non-B DNA are important for triplet repeats (Fig. The structure with duplex and CTG sequences is by these triplet repeat sequences. that the is a for Slipped structures are the only for the observed mismatch repair A. R. Kang S. Shimizu M. Sinden R.R. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). Hence, this may be the a non-B structure has been in vivo to its in vitro Also, hairpin may be by single-stranded regions during DNA replication (Fig. 1). Several factors the of the triplet repeat the type of sequence a with CGG the most to in E. Gellibolian, Oostra, B. A., Wells, R. D. (1996) J. Mol. Biol., in pressGoogle Scholar). the length of the repeats is important since for CGG, show a of instability with shorter inserts or This in E. coli is with the mechanism of genetic anticipation for the fragile X Y.-H. Kuhl D.P.A. A. M. Richards S. Jr., Warren S.T. Nelson D.L. Caskey C.T. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). the presence of the of triplet repeats for from human show the presence of stable and CGG triplets of similar that a other than but to the was responsible for et E.E. K. S.N. Richards C.S. Nelson D.L. Nat. Genet. 1994; PubMed Scopus Google Scholar) found that lengths of showed of repeat that the of the is an important event in the generation of to the fragile X the orientation of the insert relative to the replication origin was above (Fig. 1). the of E. coli used as host are E. coli was the for the CGG triplet repeats of to 160 repeats in plasmids with and containing than 160 CGG repeats were in and were to to smaller Hence, the vector of is the of the insert in the vector is important and may to the pausing observed at the DNA polymerase S. Ohshima K. Shimizu M. Amirhaeri S. Wells R.D. J. Biol. Chem. 1995; 270: 27014-27021Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). the number of the vector may be has been made in the in understanding hereditary diseases, but the molecular basis of genetic instabilities of triplet repeats to be The of expansion systems for molecular and genetic The of a is DNA or its structures may be Hence, is not surprising that this a number of other diseases also show anticipation, the may be in its In the these issues a for a of human transgenic as well as physical The is to the molecular mechanisms responsible for genetic instabilities and to these human neuromuscular and neurodegenerative diseases. for and that made this possible and R. for in of