[show abstract][hide abstract] ABSTRACT: Flap endonuclease 1 (FEN1) is a central component of Okazaki fragment maturation in eukaryotes. Genetic analysis of Saccharomyces cerevisiae FEN1 (RAD27) also reveals its important role in preventing trinucleotide repeat (TNR) expansion. In humans such expansion is associated with neurodegenerative diseases. In vitro, FEN1 can inhibit TNR expansion by employing its endonuclease activity to compete with DNA ligase I. Here we employed two yeast FEN1 nuclease mutants, rad27-G67S and rad27-G240D, to further define the mechanism by which FEN1 prevents TNR expansion. Using a yeast artificial chromosome system that can detect both TNR instability and fragility, we demonstrate that the G240D but not the G67S mutation increases both the expansion and fragility of a CTG tract in vivo. In vitro, the G240D nuclease is proficient in cleaving a fixed nonrepeat double flap; however, it exhibits severely impaired cleavage of both nonrepeat and CTG-containing equilibrating flaps. In contrast, wild-type FEN1 and the G67S mutant exhibit more efficient cleavage on an equilibrating flap than on a fixed CTG flap. The degree of TNR expansion and the amount of chromosome fragility observed in the mutant strains correlate with the severity of defective flap cleavage in vitro. We present a model to explain how flap equilibration and the unique tracking mechanism of FEN1 can collaborate to remove TNR flaps and prevent repeat expansion.
Molecular and Cellular Biology 06/2004; 24(9):4049-64. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Short DNA segments designated Okazaki fragments are intermediates in eukaryotic DNA replication. Each contains an initiator RNA/DNA primer (iRNA/DNA), which is converted into a 5'-flap and then removed prior to fragment joining. In one model for this process, the flap endonuclease 1 (FEN1) removes the iRNA. In the other, the single-stranded binding protein, replication protein A (RPA), coats the flap, inhibits FEN1, but stimulates cleavage by the Dna2p helicase/nuclease. RPA dissociates from the resultant short flap, allowing FEN1 cleavage. To determine the most likely process, we analyzed cleavage of short and long 5'-flaps. FEN1 cleaves 10-nucleotide fixed or equilibrating flaps in an efficient reaction, insensitive to even high levels of RPA or Dna2p. On 30-nucleotide fixed or equilibrating flaps, RPA partially inhibits FEN1. CTG flaps can form foldback structures and were inhibitory to both nucleases, however, addition of a dT(12) to the 5'-end of a CTG flap allowed Dna2p cleavage. The presence of high Dna2p activity, under reaction conditions favoring helicase activity, substantially stimulated FEN1 cleavage of tailed-foldback flaps and also 30-nucleotide unstructured flaps. Our results suggest Dna2p is not used for processing of most flaps. However, Dna2p has a role in a pathway for processing structured flaps, in which it aids FEN1 using both its nuclease and helicase activities.
Journal of Biological Chemistry 05/2004; 279(15):15014-24. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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. Annealing 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. We 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 upstream 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, allowed expansion as long as each loop was nine 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 propose 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 suggests that the equilibrium concentration of double loop intermediates is so vanishingly small that they are not likely contributors to sequence expansion.
Journal of Biological Chemistry 11/2003; 278(44):42854-66. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Repeat sequences in various genomes undergo expansion by poorly understood mechanisms. By using an oligonucleotide system containing such repeats, we recapitulated the last steps in Okazaki fragment processing, which have been implicated in sequence expansion. A template containing either triplet or tandem repeats was annealed to a downstream primer containing complementary repeats at its 5'-end. Overlapping upstream primers, designed to strand-displace varying numbers of repeats in the downstream primer, were annealed. Human DNA ligase I joined overlapping segments of repeats generating an expansion product from the primer strands. Joining efficiency decreased with repeat length. Flap endonuclease 1 (FEN1) cleaved the displaced downstream strand and together with DNA ligase I produced non-expanded products. However, both expanded and non-expanded products formed irrespective of relative nuclease and ligase concentrations tested or enzyme addition order, suggesting the pre-existence and persistence of intermediates leading to both outcomes. FEN1 activity decreased with the length of repeat segment displaced presumably because the flap forms structures that inhibit cleavage. Increased MgCl(2) disfavored ligation of substrate intermediates that result in expansion products. Examination of expansion in vitro enables dissection of substrate and replication enzyme dynamics on repeat sequences.
Journal of Biological Chemistry 07/2002; 277(25):22361-9. · 4.65 Impact Factor