Inhibition of flap endonuclease 1 by flap secondary structure and relevance to repeat sequence expansion

Recent genetic evidence indicates that null mutants of the 5'-flap endonucl ease (FEN1) result in an expansion of repetitive sequences. The substrate f or FEN1 is a flap formed by natural 5'-end displacement of the short interm ediates of lagging strand replication, FEN1 binds the 5'-end of the flap, t racks to the point of annealing at the base of the flap, and then cleaves. Here Re examine mechanisms by which foldback structures within the flap cou ld contribute to repeat expansions. Cleavage by FEN1 was reduced with incre ased length of the foldback. However, even the longest foldbacks were cleav ed at a low rate. Substrates containing the repetitive sequence CTG also we re cleaved at a reduced rate, Bubble substrates, likely intermediates in re peat expansions, were inhibitory. Neither replication protein A nor prolife rating cell nuclear antigen were able to assist in the removal of secondary structure within a flap. We propose that FEN1 cleaves natural foldbacks at a reduced rate. However, although the cleavage delay is not likely to infl uence the overall process of chromosomal replication, specific foldbacks co uld inhibit cleavage sufficiently to result in duplication of the foldback sequence.