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Papers In Press, published online ahead of print August 5, 2003
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642
Corresponding Author: robert_bambara{at}urmc.rochester.edu
We previously developed a system to investigate the mechanism of repeat sequence expansion during eukaryotic Okazaki fragment processing. Upstream and downstream primers were annealed to a complementary template to overlap across a CAG repeat region. An nealing by the competing primers lead to structural intermediates that ligated to expand the repeat segment. When an equal number of repeats overlapped on the upstream and downstream primers, a 2-fold expansion was expected, but no expansion occurred. W e show here that such substrates do not expand irrespective of their repeat length. To reveal mechanism, we tested different hairpin loop intermediates expected to form and facilitate ligation. Substrates configured to form large loops in either the ups tream or downstream primer alone, allowed expansion. Large or small fixed position single loops allowed expansion when located at least six nucleotides up- or downstream of the nick. Fixed loops in both primers, simulating a double loop intermediate, all owed expansion as long as each loop was 9 nucleotides from the nick. Thus neither the double-loop configuration required to form with equal length overlaps nor the large single loop configuration are fundamental structural impediments to expansion. We p ropose a model for the expansion mechanism based on the relative stabilities of single loop, double loop, hairpin and flap intermediates that is consistent with the observed expansion efficiency of equal and unequal overlap substrates. The model sug gests that the equilibrium concentration of double loop intermediates is so vanishingly small that they are not likely contributors to sequence expansion. ‰
J. Biol. Chem, 10.1074/jbc.M305137200
Submitted on May 16, 2003
Revised on August 1, 2003
Accepted on August 4, 2003
Analysis of DNA replication intermediates suggests mechanisms of repeat sequence expansion
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