DNA Ligase I Competes with FEN1 to Expand Repetitive DNA Sequences in Vitro

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 preexistence 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.Sites in which mono-to pentanucleotide DNA sequences are repeated are widespread in the chromosomes of organisms from bacteria to humans. These microsatellite repeats range from tens to hundreds of base pairs in length (1). Typically repeat sequences are highly polymorphic, and the number of repeats at specific sites varies extensively. These segments of repeats are inherently unstable and undergo sequence expansions and deletions. Especially interesting are triplet repeat sequences, because only 3 of the 10 possible triplet repeats (CAG, CGG, and GAA) are prone to expansion (2). Propensity for expansion generally increases with the size of the repeat region, and expansion can range from addition of a few repeats to thousands (3). In addition, the rate and size of sequence expansions vary between and within tissues of organisms (4). Mechanisms of sequence expansion have attracted much attention because of the involvement of triplet repeat expansion in the pathogenesis of at least 11 neurological disorders (3).Genetic studies in yeast and E. coli suggest that one mechanism of expansion involves slipped mispairing of template and primer strands within the repeat region during DNA replication or repair (5, 6). Such mispairing results in formation of a loop in one strand of the helix. Looping of the template strand during replication of the leading or lagging strand would lead to deletion, whereas that in the primer strand would result in expansion. Triplet repeats have been shown to be inherently flexible and mispair to form slipped DNA intermediates (7,8). Additionally, the quasi-palindromic nature of triplet repeats results in the f...

Haploinsufficiency of yeast FEN1 causes instability of expanded CAG/CTG tracts in a length-dependent manner

“…However, when Fen1 concentration is limiting, the unprocessed flaps could equilibrate into a variety of intermediate structures, some of which will be ligated by DNA ligase to cause a sequence expansion. This model is supported by in vitro data that shows that there is a competition between Fen1 and DNA ligase at the flap, with increasing amounts of ligase resulting in ligation of the unprocessed flaps to produce expansions (Henricksen et al, 2002). Similarly, limiting amount of Fen1 could also favor flap ligation to produce expansions.…”

Interactions Among DNA Ligase I, the Flap Endonuclease and Proliferating Cell Nuclear Antigen in the Expansion and Contraction of CAG Repeat Tracts in Yeast

“…In one study, DNA ligase I and the flap endonuclease have been allowed to compete on a substrate containing a flap of CTG repeats (Henricksen et al 2002). This study shows that in a competition between the two enzymes, DNA ligase trumps the flap endonuclease in capturing bubbles and creating expanded tracts.…”

Hairpin Structure-forming Propensity of the (CCTG·CAGG) Tetranucleotide Repeats Contributes to the Genetic Instability Associated with Myotonic Dystrophy Type 2

“…However, because of the singlestranded nature of the Okazaki fragments on the lagging strand, both expansions and deletions can occur depending on the stability of the hairpin structures formed by these repeats. The aberrant processing of the Okazaki fragments involving FEN-1 and DNA ligase has also been hypothesized to play an important role in generating genetic instabilities (21,(113)(114)(115)(116)(117)(118).…”