Nucleic Acids Research Advance Access published online on November 16, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl846
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Molecular Biology |
Replication in mammalian cells recapitulates the locus-specific differences in somatic instability of genomic GAA triplet-repeats
1 Department of Biochemistry and Molecular Biology Oklahoma City, OK 73104, USA 2 Department of Pediatrics, University of Oklahoma Health Sciences Center Oklahoma City, OK 73104, USA
*To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 975 NE, 10th, BRC458, Oklahoma City, OK 73104, USA. Tel: +1 405 271 1360; Fax: +1 405 271 3910; Email: Sanjay-Bidichandani{at}ouhsc.edu
Received July 24, 2006. Revised October 9, 2006. Accepted October 9, 2006.
Friedreich ataxia is caused by an expanded (GAA·TTC)n sequence in intron 1 of the FXN gene. Small pool PCR analysis showed that pure (GAA·TTC)44+ sequences at the FXN locus are unstable in somatic cells in vivo, displaying both expansions and contractions. On searching the entire human and mouse genomes we identified three other genomic loci with pure (GAA·TTC)44+ sequences. Alleles at these loci showed mutation loads of <1% compared with 6.3–30% for FXN alleles of similar length, indicating that somatic instability in vivo is regulated by locus-specific factors. Since distance between the origin of replication and the (CTG·CAG)n sequence modulates repeat instability in mammalian cells, we tested if this could also recapitulate the locus-specific differences for genomic (GAA·TTC)n sequences. Repeat instability was evaluated following replication of a (GAA·TTC)115 sequence in transfected COS1 cells under the control of the SV40 origin of replication located at one of five different distances from the repeat. Indeed, depending on the location of the SV40 origin relative to the (GAA·TTC)n sequence, we noted either no instability, predominant expansion or both expansion and contraction. These data suggest that mammalian DNA replication is a possible mechanism underlying locus-specific differences in instability of GAA triplet-repeat sequences.
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