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BIBTEX RIS

Gene Conversion (Recombination) Mediates Expansions of CTG·CAG Repeats

2000; Elsevier BV; Volume: 275; Issue: 51 Linguagem: Inglês

10.1074/jbc.m007153200

ISSN

1083-351X

Autores

John P. Jakupciak, Robert D. Wells,

Tópico(s)

Metabolism and Genetic Disorders

Resumo

Genetic recombination is a robust mechanism for expanding CTG·CAG triplet repeats involved in the etiology of hereditary neurological diseases (Jakupciak, J. P., and Wells, R. D. (1999) J. Biol. Chem. 274, 23468–23479). This two-plasmid recombination system in Escherichia coli with derivatives of pUC19 and pACYC184 was used to investigate the effect of triplet repeat orientation on recombination and extent of expansions; tracts of 36, 50, 80, and 36, 100, and 175 repeats in length, respectively, in all possible permutations of length and in both orientations (relative to the unidirectional replication origins) revealed little or no effect of orientation of expansions. The extent of expansions was generally severalfold the length of the progenitor tract and frequently exceeded the combined length of the two tracts in the cotransformed plasmids. Expansions were much more frequent than deletions. Repeat tracts bearing two G-to-A interruptions (polymorphisms) within either 171- or 219-base pair tracts substantially reduced the expansions compared with uninterrupted repeat tracts of similar lengths. Gene conversion, rather than crossing over, was the recombination mechanism. Prior studies showed that DNA replication, repair, and tandem duplication also mediated genetic instabilities of the triplet repeat sequence. However, gene conversion (recombinational repair) is by far the most powerful expansion mechanism. Thus, we propose that gene conversion is the likely expansion mechanism for myotonic dystrophy, spinocerebellar ataxia type 8, and fragile X syndrome. Genetic recombination is a robust mechanism for expanding CTG·CAG triplet repeats involved in the etiology of hereditary neurological diseases (Jakupciak, J. P., and Wells, R. D. (1999) J. Biol. Chem. 274, 23468–23479). This two-plasmid recombination system in Escherichia coli with derivatives of pUC19 and pACYC184 was used to investigate the effect of triplet repeat orientation on recombination and extent of expansions; tracts of 36, 50, 80, and 36, 100, and 175 repeats in length, respectively, in all possible permutations of length and in both orientations (relative to the unidirectional replication origins) revealed little or no effect of orientation of expansions. The extent of expansions was generally severalfold the length of the progenitor tract and frequently exceeded the combined length of the two tracts in the cotransformed plasmids. Expansions were much more frequent than deletions. Repeat tracts bearing two G-to-A interruptions (polymorphisms) within either 171- or 219-base pair tracts substantially reduced the expansions compared with uninterrupted repeat tracts of similar lengths. Gene conversion, rather than crossing over, was the recombination mechanism. Prior studies showed that DNA replication, repair, and tandem duplication also mediated genetic instabilities of the triplet repeat sequence. However, gene conversion (recombinational repair) is by far the most powerful expansion mechanism. Thus, we propose that gene conversion is the likely expansion mechanism for myotonic dystrophy, spinocerebellar ataxia type 8, and fragile X syndrome. triplet repeat sequence(s) base pair(s) Several hereditary neurological diseases including myotonic dystrophy, fragile X syndrome, spinocerebellar ataxia type 8, and Friedreich's ataxia result from expanded TRS1 CTG·CAG, CGG·CCG, and GAA·TTC within or near their genes (reviewed in Ref. 1Wells, R. D., and Warren, S. T. (eds) (1998) Genetic Instabilities and Hereditary Neurological Diseases, Academic Press, Inc., San DiegoGoogle Scholar). For these diseases, the TRS expansions occur by hundreds of repeats and can appear rapidly within a pedigree. However, for other diseases (Huntington's disease, spinocerebellar ataxia type 1, and Kennedy's disease), the CAG·CTG repeats expand to a smaller extent (tens of repeats) and occur in exons and, hence, lengthen the oligoglutamine tracts in the relevant proteins. The earlier age of onset and the increased severity of most of these neurological diseases in successive generations (clinically referred to as anticipation) are correlated to the lengths of the TRS. Long tracts of TRS are unstable and show repeat size polymorphisms in successive generations and in different tissues. In addition to these observations in humans, the molecular mechanisms of TRS instabilities have been investigated in Escherichia coli (2–14), yeast (15Freudenreich C.H. Kantrow S.M. Zakian V.A. Science. 1998; 279: 853-856Crossref PubMed Scopus (360) Google Scholar, 16Schweitzer J.K. Livingston D.M. Hum. Mol. Genet. 1997; 6: 349-355Crossref PubMed Scopus (87) Google Scholar, 17Kokoska R.J. Stefanovic L. Tran H.T. 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Skinner P.J. Kaytor M.D. Yi H. Hersch S.M. Clark H.B. Zoghbi H.Y. Orr H.T. Cell. 1998; 95: 41-53Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar, 25Mangiarine L. Sathasivam K. Majal A. Mott R. Seller M. Bates G.P. Nat. Genet. 1997; 15: 197-200Crossref PubMed Scopus (259) Google Scholar, 26Cummings C.J. Orr H.T. Zoghbi H.Y. Philos. Trans. R. Soc. Lond-Biol. Sci. 1999; 354: 1079-1081Crossref PubMed Scopus (37) Google Scholar).The molecular mechanisms of genetic instability of TRS have been intensively studied because of their pivotal role in the disease pathogenesis. A number of investigations have revealed the involvement of misalignment-mediated DNA synthesis (1–16). These expansions and deletions are thought to be due to the formation of unusual DNA secondary structures and slipped complementary strands at polymerase pause sites that cause frameshift mutations during DNA synthesis (8Ohshima K. Kang S. Larson J.E. Wells R.D. J. Biol. Chem. 1996; 271: 16773-16783Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13Ohshima K. Wells R.D. J. Biol. Chem. 1997; 272: 16798-16806Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 27Samadashwily G.M. Raca G. Mirkin S.M. Nat. Genet. 1997; 17: 298-304Crossref PubMed Scopus (284) Google Scholar). Related studies revealed the involvement of nucleotide excision repair (11Parniewski P. Bacolla A. Jaworski A. Wells R.D. Nucleic Acids Res. 1999; 27: 616-623Crossref PubMed Scopus (79) Google Scholar), methyl-directed mismatch repair (6Jaworski A. Rosche W.A. Gellibolian R. Kang S. Shimizu M. Bowater R.P. Sinden R.R. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11019-11023Crossref PubMed Scopus (147) Google Scholar, 12Wells R.D. Parniewski P. Pluciennik A. Bacolla A. Gellibolian R. Jaworski A. J. Biol. Chem. 1998; 273: 19532-19541Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 28Schumacher S. Fuchs R.P.P. Bichara M. J. Mol. Biol. 1998; 279: 1101-1110Crossref PubMed Scopus (56) Google Scholar), DNA polymerase III proofreading (14Iyer R.R. Pluciennik A. Rosche W.A. Sinden R.R. Wells R.D. J. Biol. Chem. 2000; 275: 2174-2184Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), DNA damage (abasic sites) repair (29Darden-Lyons T. Topal M.D. J. Biol. Chem. 1999; 274: 25975-25978Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), flap endonuclease (30Richard G.F. Dujon B. Haber J.E. Mol. Gen. Genet. 1999; 26: 871-882Crossref Scopus (85) Google Scholar), and transcription (7Bowater R.P. Jaworski A. Larson J.E. Parniewski P. Wells R.D. Nucleic Acids Res. 1997; 25: 2861-2868Crossref PubMed Scopus (86) Google Scholar). However, the possible participation of genetic recombination in expansion has been less clear.Several human genetic studies on patient materials reported haplotype analyses, especially related to myotonic dystrophy (31Tsilfidis D. MacKenzie A.E. Mettler G. 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Mulley J. Sutherland G.R. J. Med. Genet. 1992; 28: 818-823Crossref Scopus (104) Google Scholar), unequal crossing over as a general mechanism for expansion has not been favored. Alternatively, CAG·CTG repeats were reported to be hot spots for recombination (40Szemraj J. Plucienniczak G. Jaworski J. Plucienniczak A. Gene (Amst. ). 1995; 152: 261-264Crossref PubMed Scopus (57) Google Scholar), and recent experiments in yeast suggested that (CTG·CAG) (15Freudenreich C.H. Kantrow S.M. Zakian V.A. Science. 1998; 279: 853-856Crossref PubMed Scopus (360) Google Scholar) and (CGG·CCG) (19Balakumaran B.S. Freudenreich C.H. Zakian V.A. Hum. Mol. Genet. 2000; 9: 93-100Crossref PubMed Scopus (73) Google Scholar) repeats are susceptible to strand breaks. Yeast rad27 strains had augmented instability of the TRS. It is known that the majority of errors that accumulate inrad27 strains are processed via single strand annealing as well as double-stranded break repair (41Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar), which proceeds via gene conversion (42Richardson C. Moynahan M.E. Jasin M. Genes Dev. 1998; 12: 3831-3842Crossref PubMed Scopus (338) Google Scholar, 43Lin Y. Lukacsovich T. Waldman A.S. Mol. Cell. Biol. 1999; 19: 8353-8360Crossref PubMed Scopus (72) Google Scholar).To evaluate the potential role of genetic recombination (via gene conversion or unequal crossing over) as a mechanism for expansions of CTG·CAG repeats, we established (44Jakupciak J.P. Wells R.D. J. Biol. Chem. 1999; 274: 23468-23479Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) an in vivorecombination system in E. coli. The expansion events were dependent on the presence of long CTG·CAG sequences in the two-plasmid recombination system and required recombination-proficient cells to give frequent, severalfold expansions. Recombination was proven genetically and biochemically. Thus, it was concluded that if these reactions occur in humans, recombination may contribute, along with replication and repair, to the expansions responsible for anticipation associated with the hereditary neurological syndromes. Herein, we describe studies on the molecular mechanism of the recombination process (gene conversion or unequal crossing over) and the lack of effect of TRS orientation on the multiple fold expansions.DISCUSSIONThis report describes the direct demonstration that gene conversion mediates the expansions of CTG·CAG 3For the sake of brevity, CTG·CAG refers under "Discussion" to either CTG·CAG or CAG·CTG. repeats. Gene conversion is the principal recombination mechanism rather than unequal crossing over. Also, the results from a large number of gene conversion-mediated expansion studies with plasmids containing TRS inserts in all possible permutations of the orientations revealed little or no influence of orientation. Hence, these data provide further verification that recombination (not complementary strand slippage at the replication fork) is the responsible mechanism. Gene conversion (recombinational repair) is the nonreciprocal transfer of genetic information from one DNA duplex to another with no exchange of flanking sequences (55Szostak J.W. Orr-Weaver R.L. Rothstein R.J. Cell. 1983; 33: 25-35Abstract Full Text PDF PubMed Scopus (1745) Google Scholar, 56Cox M.M. Genes Cells. 1998; 3: 65-78Crossref PubMed Scopus (103) Google Scholar, 57West S.C. Annu. Rev. Biochem. 1992; 61: 603-640Crossref PubMed Scopus (302) Google Scholar). Hence, this would explain the linkage disequilibrium of flanking markers in haplotype analyses (1Wells, R. D., and Warren, S. T. (eds) (1998) Genetic Instabilities and Hereditary Neurological Diseases, Academic Press, Inc., San DiegoGoogle Scholar, 52Kunst C.B. Warren S.T. Cell. 1994; 77: 853-861Abstract Full Text PDF PubMed Scopus (320) Google Scholar, 53Eichler E.E. Holden J.J.A. Popovich B.W. Reiss A.L. Snow K. Thibodeau S.N. Richards C.S. Ward P.A. Nelson D.L. Nat. Genet. 1994; 8: 88-94Crossref PubMed Scopus (413) Google Scholar, 54Kunst C.B. Zerylnick C. Karickhoff L. Eichler E. Bullard J. Chalifoux M. Holden J.J. Torroni A. Nelson D.L. Warren S.T. Am. J. Hum. Genet. 1996; 58: 513-522PubMed Google Scholar), if this recombination mechanism is also responsible for expansions in human cells. Whereas our paper (44Jakupciak J.P. Wells R.D. J. Biol. Chem. 1999; 274: 23468-23479Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) was the first to demonstrate the involvement of recombination in TRS expansions, other workers (58Gangloff S. Zou H. Rothstein R. EMBO J. 1996; 15: 1715-1725Crossref PubMed Scopus (114) Google Scholar) demonstrated that gene conversion plays the major role in controlling the instability of large tandem repeats of ribosomal DNA sequences in yeast. Although the ribosomal tandem repeat sequences are much longer than triplets, the expansion mechanism is probably the same.The gene conversion (recombinational repair) mechanism responsible for the repeat tract expansions is modeled in Fig. 3. The DNA replication fork stalls when it encounters a CTG·CAG sequence (8Ohshima K. Kang S. Larson J.E. Wells R.D. J. Biol. Chem. 1996; 271: 16773-16783Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13Ohshima K. Wells R.D. J. Biol. Chem. 1997; 272: 16798-16806Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 27Samadashwily G.M. Raca G. Mirkin S.M. Nat. Genet. 1997; 17: 298-304Crossref PubMed Scopus (284) Google Scholar), which can result in double-stranded DNA breaks that widen into double-stranded gaps (41Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 56Cox M.M. Genes Cells. 1998; 3: 65-78Crossref PubMed Scopus (103) Google Scholar, 59Harmon F.G. Kowalczykowski S.C. Genes Dev. 1998; 12: 1134-1144Crossref PubMed Scopus (235) Google Scholar). When breaks occur in the repeat tract (15Freudenreich C.H. Kantrow S.M. Zakian V.A. Science. 1998; 279: 853-856Crossref PubMed Scopus (360) Google Scholar, 19Balakumaran B.S. Freudenreich C.H. Zakian V.A. Hum. Mol. Genet. 2000; 9: 93-100Crossref PubMed Scopus (73) Google Scholar) of the progenitor, the resultant gap may range in size from 40 to 400 repeats (55Szostak J.W. Orr-Weaver R.L. Rothstein R.J. Cell. 1983; 33: 25-35Abstract Full Text PDF PubMed Scopus (1745) Google Scholar, 60Cordeiro-Stone M. Makhov A.M. Zaritskaya L.S. Griffith J.D. J. Mol. Biol. 1999; 289: 1207-1218Crossref PubMed Scopus (85) Google Scholar). A search for homology then takes place, and the ends of the progenitor strands invade the TRS in the pACYC184 derivative (Fig. 3,upper right panel). After annealing, the gap is filled in by recombination repair and ligated to finalize the expansion of the TRS tract in the pUC19 derivative. In addition to these standard recombination repair steps, it is possible with the triplet repeat tracts that DNA slippage may enable the formation of hairpin structures (center, right) (2–13, 21Maurer D.J. O'Callaghan B.L. Livingston D.M. Mol. Cell. Biol. 1996; 16: 6617-6622Crossref PubMed Scopus (108) Google Scholar, 24Klement I.A. Skinner P.J. Kaytor M.D. Yi H. Hersch S.M. Clark H.B. Zoghbi H.Y. Orr H.T. Cell. 1998; 95: 41-53Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar, 25Mangiarine L. Sathasivam K. Majal A. Mott R. Seller M. Bates G.P. Nat. Genet. 1997; 15: 197-200Crossref PubMed Scopus (259) Google Scholar, 26Cummings C.J. Orr H.T. Zoghbi H.Y. Philos. Trans. R. Soc. Lond-Biol. Sci. 1999; 354: 1079-1081Crossref PubMed Scopus (37) Google Scholar, 61Usdin K. Woodford K.J. Nucleic Acids Res. 1995; 23: 4202-4209Crossref PubMed Scopus (224) Google Scholar, 62Koob J.D. Moseley M.L. Schut L.J. Benzow K.A. Bird T.D. Day J.W. Ranum L.P.W. Nat. Genet. 1999; 21: 379-383Crossref PubMed Scopus (537) Google Scholar, 63Mitas M., Yu, A. Dill J. Haworth I.S. Biochemistry. 1995; 34: 12803-12811Crossref PubMed Scopus (112) Google Scholar, 64Gacy A.M. Goellner G. Juranic N. Macura S. McMurray C.T. Cell. 1995; 81: 533-540Abstract Full Text PDF PubMed Scopus (515) Google Scholar). Thus, these two processes may act in concert to enhance the formation of expansions. Others have suggested that gene conversion and break-induced replication are cooperative processes (65Haber J.E. Trends Biochem. Sci. 1999; 24: 271-275Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Because double-stranded breaks occur frequently under normal growth conditions and recBCD and recA proteins are essential for recombinational repair (55Szostak J.W. Orr-Weaver R.L. Rothstein R.J. Cell. 1983; 33: 25-35Abstract Full Text PDF PubMed Scopus (1745) Google Scholar, 66Michel B. Ehrlich S.D. Uzest M. EMBO J. 1997; 16: 430-438Crossref PubMed Scopus (379) Google Scholar, 67Uzest M. Ehrlich S.D. Michel B. Mol. Microbiol. 1995; 17: 1177-1188Crossref PubMed Scopus (67) Google Scholar), this also suggests a coupling of replication and recombination.Our data (Fig. 2 and Table I) demonstrate that gene conversion mediates the multiple fold expansions of CTG·CAG sequences, irrespective of the orientation of the TRS tracts. These results are in stark contrast to some prior studies in E. coli (2–13) and yeast (15Freudenreich C.H. Kantrow S.M. Zakian V.A. Science. 1998; 279: 853-856Crossref PubMed Scopus (360) Google Scholar, 16Schweitzer J.K. Livingston D.M. Hum. Mol. Genet. 1997; 6: 349-355Crossref PubMed Scopus (87) Google Scholar, 68Miret J.J. Pesson-Brandao L.P. Lahue R.S. Mol. Cell. Biol. 1997; 17: 3382-3387Crossref PubMed Scopus (82) Google Scholar), which demonstrated a marked influence of the orientation of the repeat tracts on deletions and expansions. These results were explained by slippage of the complementary repeat tracts at the replication fork, which was enhanced by the preferential formation of DNA looped conformations (13Ohshima K. Wells R.D. J. Biol. Chem. 1997; 272: 16798-16806Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 63Mitas M., Yu, A. Dill J. Haworth I.S. Biochemistry. 1995; 34: 12803-12811Crossref PubMed Scopus (112) Google Scholar, 64Gacy A.M. Goellner G. Juranic N. Macura S. McMurray C.T. Cell. 1995; 81: 533-540Abstract Full Text PDF PubMed Scopus (515) Google Scholar, 69Pearson C.E. Sinden R.R. Biochemistry. 1996; 35: 5041-5053Crossref PubMed Scopus (229) Google Scholar) that enabled bypass synthesis at the replication fork. When CTG repeats were in the lagging strand template, deletions were more likely than expansions. This behavior could also occur on the leading strand template (49Iyer R.R. Wells R.D. J. Biol. Chem. 1999; 274: 3865-3877Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). However, in the studies described herein in recombination-proficient cells with the two-plasmid system, the inversion of the repeat tract does not cause an alteration in the instability, as expected from recombination mechanisms (55Szostak J.W. Orr-Weaver R.L. Rothstein R.J. Cell. 1983; 33: 25-35Abstract Full Text PDF PubMed Scopus (1745) Google Scholar, 57West S.C. Annu. Rev. Biochem. 1992; 61: 603-640Crossref PubMed Scopus (302) Google Scholar, 65Haber J.E. Trends Biochem. Sci. 1999; 24: 271-275Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 70Cohen H. Sears D.D. Zenvirth D. Hieter P. Simchen G. Mol. Cell. Biol. 1999; 19: 4153-4158Crossref PubMed Scopus (29) Google Scholar). Recombinational repair (Fig. 3) is dependent on sequence homology but not sequence orientation. Hence, these results further verify that recombination is the mechanism involved in the multiple fold expansion process. Although gene conversion is the simplest mechanism to explain our data, we cannot eliminate the possibility of a double unequal cross-over event. Also, we demonstrated the requirement for recA as well as recBC functions. As shown in Fig. 3, recombination and DNA synthetic processes may act in concert to generate the instabilities.The lengths of the repeat tracts as well as the absence or the presence of interruptions influences the expansion process. In our two-plasmid recombination system, the pACYC184 derivatives containing only 36 or 39 repeats were unable to effect expansions, whereas 100 or 175 repeats were extremely active, regardless of the length of the TRS in the pUC19 derivatives. Hence, this effect of length is consistent with prior reports (57West S.C. Annu. Rev. Biochem. 1992; 61: 603-640Crossref PubMed Scopus (302) Google Scholar) on recombination mechanisms and may explain why other workers observed only deletions in their yeast recombination system (30Richard G.F. Dujon B. Haber J.E. Mol. Gen. Genet. 1999; 26: 871-882Crossref Scopus (85) Google Scholar).A hallmark of triplet repeat diseases is that intermediate size repeats lose a snip (single nucleotidepolymorphism) and subsequently expand by severalfold (52Kunst C.B. Warren S.T. Cell. 1994; 77: 853-861Abstract Full Text PDF PubMed Scopus (320) Google Scholar, 53Eichler E.E. Holden J.J.A. Popovich B.W. Reiss A.L. Snow K. Thibodeau S.N. Richards C.S. Ward P.A. Nelson D.L. Nat. Genet. 1994; 8: 88-94Crossref PubMed Scopus (413) Google Scholar, 54Kunst C.B. Zerylnick C. Karickhoff L. Eichler E. Bullard J. Chalifoux M. Holden J.J. Torroni A. Nelson D.L. Warren S.T. Am. J. Hum. Genet. 1996; 58: 513-522PubMed Google Scholar). Without the loss of the interruption, the repeat is stably transmitted through a pedigree. We compared the effect of interruptions on gene conversion-mediated expansions with sequences containing no interruptions. However, when the interrupted pUC19 derivatives (pRW3753 and pRW3755) were analyzed, few or no expansions occurred. These plasmids contain two G-to-A point mutations; thus, if the complementary strands slip relative to each other, four mismatches will be generated. Prior work demonstrated that these mismatches effectively destabilize the formation of slipped structures (69Pearson C.E. Sinden R.R. Biochemistry. 1996; 35: 5041-5053Crossref PubMed Scopus (229) Google Scholar). Thus, the polymerase complex is not likely to pause, and the replication fork will not collapse which will limit the number of double-stranded breaks that are inflicted. Whereas four mismatches in 219 bp is a small percentage, this extent of heterology (1.8%) was quite effective in eliminating the formation of multiple fold expansion products in our system. Similarly, other workers have recently demonstrated that as little as 1.2% heterology in a mammalian cell recombination system reduces the effects of recombination (71Elliott B. Richardson C. Winderbaum J. Nickoloff J.A. Jasin M. Mol. Cell. Biol. 1998; 18: 93-101Crossref PubMed Scopus (258) Google Scholar).Little or no correlation was found between the length of the CTG·CAG tracts in pACYC184 (100 and 175 repeats in length) and pUC19 derivatives with the size of the expansion products. Specifically, any individual clone from a single transformation may contain products of many different sizes. Some clones had only one expansion product, and others had several products. A few clones had both expansions and deletions of the TRS. However, the lengths of these instabilities were essentially random. Interestingly, the repeat tracts were expanded to generate discrete lengths rather than smears representing a large number of related-length molecules. The reason for this behavior is uncertain and is under further investigation. Furthermore, the frequency of expansions to deletions in this gene conversion system is extremely high (approximately 1.5 × 103-fold). This behavior is in marked contrast to the prior investigations in recombination-deficient E. coli and in yeast where expansions were substantially less frequent than deletions by a ratio of approximately 1:100 (2–13, 15Freudenreich C.H. Kantrow S.M. Zakian V.A. Science. 1998; 279: 853-856Crossref PubMed Scopus (360) Google Scholar, 16Schweitzer J.K. Livingston D.M. Hum. Mol. Genet. 1997; 6: 349-355Crossref PubMed Scopus (87) Google Scholar, 68Miret J.J. Pesson-Brandao L.P. Lahue R.S. Mol. Cell. Biol. 1997; 17: 3382-3387Crossref PubMed Scopus (82) Google Scholar). Thus, we believe that powerful recombination processes may be responsible for the large expansions that are observed in human genetic studies of myotonic dystrophy, spinocerebellar ataxia type 8, Friedreich's ataxia, and fragile X syndrome (reviewed in Ref. 1Wells, R. D., and Warren, S. T. (eds) (1998) Genetic Instabilities and Hereditary Neurological Diseases, Academic Press, Inc., San DiegoGoogle Scholar).In summary, gene conversion is a robust mechanism for effecting multiple fold expansions of CTG·CAG tracts in this genetically and biochemically tractable system. Of course, it is desirable to extend these investigations into human systems to evaluate its role in disease pathogenesis. The development of a suitable human gene conversion system will be required to rigorously evaluate this question. Whereas this will present challenges, it may be noted that a recent investigation on individuals with an Ataxia-Telangiectasia-like disorder showed a link between genetic defects in double-stranded break repair genes and this disease of chromosomal instability (72Stewart S.S. Masser R.S. Stankovic T. Bressan D.A. Kaplan M.I. Jaspers N.G.N. Raams A. Byrd P.J. Petrini J.H.J. Taylor A.M.R. Cell. 1999; 99: 577-587Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar). Hence, if a link is established between gene conversion mechanisms and the expansions (anticipation) observed in certain human hereditary neurological diseases, new targets for therapeutic interventions may be established. Several hereditary neurological diseases including myotonic dystrophy, fragile X syndrome, spinocerebellar ataxia type 8, and Friedreich's ataxia result from expanded TRS1 CTG·CAG, CGG·CCG, and GAA·TTC within or near their genes (reviewed in Ref. 1Wells, R. D., and Warren, S. T. (eds) (1998) Genetic Instabilities and Hereditary Neurological Diseases, Academic Press, Inc., San DiegoGoogle Scholar). For these diseases, the TRS expansions occur by hundreds of repeats and can appear rapidly within a pedigree. However, for other diseases (Huntington's disease, spinocerebellar ataxia type 1, and Kennedy's disease), the CAG·CTG repeats expand to a smaller extent (tens of repeats) and occur in exons and, hence, lengthen the oligoglutamine tracts in the relevant proteins. The earlier age of onset and the increased severity of most of these neurological diseases in successive generations (clinically referred to as anticipation) are correlated to the lengths of the TRS. Long tracts of TRS are unstable and show repeat size polymorphisms in successive generations and in different tissues. In addition to these observations in humans, the molecular mechanisms of TRS instabilities have been investigated in Escherichia coli (2–14), yeast (15Freudenreich C.H. Kantrow S.M. Zakian V.A. Science. 1998; 279: 853-856Crossref PubMed Scopus (360) Google Scholar, 16Schweitzer J.K. Livingston D.M. Hum. Mol. Genet. 1997; 6: 349-355Crossref PubMed Scopus (87) Google Scholar, 17Kokoska R.J. Stefanovic L. Tran H.T. Resnick M.A. Gordenin D.A. Petes T.D. Mol. Cell. Biol. 1998; 18: 27

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