Triplet repeat expansion generated by DNA slippage is suppressed by human flap endonuclease 1

Human flap endonuclease 1 (h-FEN1) mutations have dramatic effects on repeat instability. Current models for repeat expansion predict that h-FEN1 protein prevents mutations by removing 5'-flaps generated at ends of Okazaki fragments by strand displacement synthesis. The models propose that hairpin formations within flaps containing repeats enable them to escape h-FEN1 cleavage. Friedreich's ataxia is caused by expansion mutations in a d(GAA)(n) repeat tract. Single-stranded d( GAA) n repeat tracts, however, do not form stable hairpins until the repeat tracts are quite long. Therefore, to understand how d( GAA) n repeat expansions survive h-FEN1 activity, we determined the effects of h-FEN1 on d( GAA) n repeat expansion during replication of a d( TTC)(n) repeat template. Replication initiated within the repeat tract generated significant expansion that was suppressed by the addition of h-FEN1 at the start of replication. The ability of h-FEN1 to suppress expansion implies that DNA slippage generates a 5'-flap in the nascent strand independent of strand displacement synthesis by an upstream polymerase. Delaying the addition of h-FEN1 to the replication reaction abolished the ability of h- FEN1 ability to suppress d(GAA)(n) repeat expansion products of all sizes, including sizes unable to hairpin. Use of model substrates demonstrated that h- FEN1 cleaves d(GAA)(n) 5'-flaps joined to double-stranded non-repeat sequences but not those joined to double-stranded repeat tracts. The results provide evidence that, given the opportunity, short d(GAA)(n) repeat expansion products rearrange from 5'-flaps to stable internal loops inside the repeat tract. Long expansion products are predicted to form hairpinned flaps and internal loops. Once formed, these DNA conformations resist hFEN1. The biological implications of the results are discussed.