In vitro (CTG)*(CAG) expansions and deletions by human cell extracts.
The mechanism of disease-associated (CTG)*(CAG) expansion may involve DNA replication slippage, replication direction, Okazaki fragment processing, recombination, or repair. A length-dependent bias for expansions is observed in humans affected by a trinucleotide repeat-associated disease. We developed an assay to test the effect of replication direction on (CTG)*(CAG) instabilities incurred during in vitro (SV40) DNA replication mediated by human cell extracts. This system recapitulates the bias for expansions observed in humans. Replication by HeLa cell extracts generated expansions and deletions that depended upon repeat tract length and the direction of replication. Templates with 79 repeats yielded predominantly expansions (CAG as lagging strand template) or predominantly deletions (CTG as lagging strand template). Templates containing 17 repeats were stable. Thus, replication direction determined the type of mutation. These results provide new insights into the orientation of replication effect upon repeat stability. This system will be useful in determining the contribution of specific human proteins to (CTG)*(CAG) expansions.
Panigrahi
Gagan B
GB
Program of Genetics and Genomic Biology, The Hospital for Sick Children, University of Toronto, Toronto, Ontario M5A 1X8, Canada.
Cleary
John D
JD
Pearson
Christopher E
CE
D015139:Blotting, Southern; D004247:DNA / Q000096:biosynthesis* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D017353:Gene Deletion; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D012150:Polymorphism, Restriction Fragment Length; D019680:Trinucleotide Repeat Expansion
D004247:DNA