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Papers In Press, published online ahead of print February 6, 2002
J. Biol. Chem, 10.1074/jbc.M109761200
Submitted on October 9, 2001
Revised on February 5, 2002
Accepted on February 5, 2002
Genetics & Genomic Biology, The Hopsital for Sick Children, Toronto, Ontario M5G 1X8
Corresponding Author: cepearson{at}genet.sickkids.on.ca
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.
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