Summary
Expansions of triplet repeats are responsible for more than 15 hereditary neurological disorders in humans (1,2). Triplet repeats are fairly stable when the number of elementary units is under approx 30, but become polymorphic in length with a clear bias for expansions when this threshold is exceeded. This results in the rapid addition of hundreds or even thousands of extra repeats and, ultimately, disease. The mechanisms of triplet repeat expansions are not yet understood. The role of several genetic processes, including replication (3), recombination (4,5), and repair (6), was suggested. However, given the swift accumulation of extra DNA material, DNA replication seems to be an intuitive candidate for generating expansions. Numerous data point to the aberrant replication of triplet repeats as a cause of triplet repeat expansions (3,7–16). Direct experimental proof of aberrant replication through triplet repeats was lacking. This encouraged us to study the mode of replication fork progression through triplet repeats in vivo. We analyzed the effects of triplet repeats on replication of bacterial or yeast plasmids using an approach called two-dimensional neutral/neutral gel electrophoresis of replication intermediates. This technique, originally developed for mapping replication origins (17,18), is also instrumental in defining replication pause sites (19). Using this technique, we were able to unambiguously demonstrate that expandable triplet repeats attenuate replication fork progression in vivo and get some insights into the mechanisms of repeat expansions (20,21).
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References
Bowater, R. P. and Wells, R. D. (2001) The intrinsically unstable life of DNA triplet repeats associated with human hereditary disorders. Prog. Nucleic Acid Res. Mol. Biol. 66, 159–202.
Siyanova, E. Y. and Mirkin, S. M. (2001) Expansion of trinucleotide repeats. Mol. Biol. (Mosc.) 35, 168–182.
Kang, S., Jaworski, A., Ohshima, K., et al. (1995) Expansion and deletion of CTG repeats from human disease genes are determined by the direction of replication in E. coli. Nature Genet. 10, 213–218.
Jakupciak, J. P. and Wells, R. D. (2000) Gene conversion (recombination) mediates expansions of CTG.CAG repeats. J. Biol. Chem. 275, 40,003–40,013.
Richard, G.-F., Goellner, G. M., McMurray, C. T., et al. (2000) Recombination-induced CAG trinucleotide repeat expansions in yeast involve the MRE11-RAD50-XRS2 complex. EMBO J. 19, 2381–2390.
Kovtun, I. V. and McMurray, C. T. (2001) Trinucleotide expansion in haploid germ cells by gap repair. Nature Genet. 27, 407–411.
Balakumaran, B. S., Freudenreich, C. H., and Zakian, V. A. (2000) CGG/CCG repeats exhibit orientation-dependent instability and orientation-independent fragility in Saccharomyces cerevisiae. Hum. Mol. Genet. 9, 93–100.
Cleary, J. D., Nichol, K., Wang, Y. H., et al. (2002) Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells. Nature Genet. 31, 37–46.
Freudenreich, C. H., Kantrow, S. M., and Zakian, V. A. (1998) Expansion and length-dependent fragility of CTG repeats in yeast. Science 279, 853–856.
Ireland, M. J., Reinke, S. S., and Livingston, D. M. (2000) The impact of lagging strand replication mutations on the stability of CAG repeat tracts in yeast. Genetics 155, 1657–1665.
Iyer, R. R., Pluciennik, A., Rosche, W. A., et al. (2000) DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli. J. Biol. Chem. 275, 2174–2184.
Miret, J. J., Pessoa-Brandao, L., and Lahue, R. S. (1998) Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 95, 12,438–12,443.
Schweitzer, J. K. and Livingston, D. M. (1999) The effect of DNA replication mutations on CAG tract stability in yeast. Genetics 152, 953–963.
Schweitzer, J. K. and Livingston, D. M. (1998) Expansions of CAG repeat tracts are frequent in a yeast mutant defective in Okazaki fragment maturation. Hum. Mol. Genet. 7, 69–74.
Spiro, C., Pelletier, R., Rolfsmeier, M. L., et al. (1999) Inhibition of FEN-1 processing by DNA secondary structure at trinucleotide repeats. Mol. Cell 4, 1079–1085.
White, P. J., Borts, R. H., and Hirst, M. C. (1999) Stability of the human fragile X (CGG)n triplet repeat array in Saccharomyces cerevisiae deficient in aspects of DNA metabolism. Mol. Cell. Biol. 19, 5675–5684.
Brewer, B. J. and Fangman, W. L. (1987) The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51, 463–471.
Huberman, J. A., Spotila, L. D., Nawotka, K. A., et al. (1987) The in vivo replication origin of the yeast 2 microns plasmid. Cell 51, 473–481.
Deshpande, A. M. and Newlon, C. S. (1996) DNA replication fork pause sites dependent on transcription. Science 272, 1030–1033.
Pelletier, R., Krasilnikova, M. M., Samadashwily, G. M., et al. (2002) Replication and expansion of trinucleotide repeats in yeast. Mol. Cell. Biol. 23, 1349–1357.
Samadashwily, G. M., Raca, G., and Mirkin, S. M. (1997) Trinucleotide repeats affect DNA replication in vivo. Nature Genet. 17, 298–304.
Martin-Parras, L., Hernandez, P., Martinez-Robles, M., et al. (1991) Unidirectional replication as visualised by two-dimensional agarose gel electrophoresis. J. Mol. Biol. 220, 843–855.
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Krasilnikova, M.M., Mirkin, S.M. (2004). Analysis of Triplet Repeat Replication by Two-Dimensional Gel Electrophoresis. In: Kohwi, Y. (eds) Trinucleotide Repeat Protocols. Methods in Molecular Biology™, vol 277. Humana Press. https://doi.org/10.1385/1-59259-804-8:019
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DOI: https://doi.org/10.1385/1-59259-804-8:019
Publisher Name: Humana Press
Print ISBN: 978-1-58829-243-8
Online ISBN: 978-1-59259-804-5
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