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 nonrepeat 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 h-FEN1. The biological implications of the results are discussed.
Ruggiero
Bethany L
BL
Lineberger Comprehensive Cancer Center, University of North Carolina Medical School, Chapel Hill, North Carolina 27599-7295, USA.
Topal
Michael D
MD
D000818:Animals; D004247:DNA / Q000737:chemistry / Q000235:genetics; D045585:Flap Endonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005621:Friedreich Ataxia / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D013379:Substrate Specificity; D018911:Trinucleotide Repeats / Q000235:genetics
D004247:DNA; D045585:Flap Endonucleases