title: Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia.
abstract: Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA*TTC)n sequences (9-105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8-107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Pollard
authors.forename: Rajesh
authors.initials: R
authors.lastname: Sharma
authors.forename: Mariluz
authors.initials: M
authors.lastname: Gómez
authors.forename: Sonali
authors.initials: S
authors.lastname: Shah
authors.forename: Martin B
authors.initials: MB
authors.lastname: Delatycki
authors.forename: Luigi
authors.initials: L
authors.lastname: Pianese
authors.forename: Antonella
authors.initials: A
authors.lastname: Monticelli
authors.forename: Bronya J B
authors.initials: BJ
authors.lastname: Keats
authors.forename: Sanjay I
authors.initials: SI
authors.lastname: Bidichandani
mesh_terms: D004247:DNA / Q000737:chemistry; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics; D005621:Friedreich Ataxia / Q000235:genetics*; D006801:Humans; D033862:Iron-Binding Proteins / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D013698:Templates, Genetic; D019680:Trinucleotide Repeat Expansion; D000096923:Frataxin
chemical_list: D004247:DNA; D033862:Iron-Binding Proteins; D000096923:Frataxin; C022262:Okazaki fragments