Inhibition of FEN-1 Processing by DNA Secondary Structure at Trinucleotide Repeats

Abstract

The mechanism by which trinucleotide expansion occurs in human genes is not understood. However, it has been hypothesized that DNA secondary structure may actively participate by preventing FEN-1 cleavage of displaced Okazaki fragments. We show here that secondary structure can, indeed, play a role in expansion by a FEN-1-dependent mechanism. Secondary structure inhibits flap processing at CAG, CGG, or CTG repeats in a length-dependent manner by concealing the 5' end of the flap that is necessary for both binding and cleavage by FEN-1. Thus, secondary structure can defeat the protective function of FEN-1, leading to site-specific expansions. However, when FEN-1 is absent from the cell, alternative pathways to simple inhibition of flap processing contribute to expansion.

Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells

“…Longer rather than shorter nascent repeat tracts at Okazaki termini might form these structures more readily. The metabolism of the structural intermediates may lead to efficient or error-prone processing by replication 22,24,[32][33][34] , repair 37,46,47 or recombination proteins [43][44][45]48 , all of which could lead to instability. Although other explanations are possible, it is clear that striking differences in (CTG)•(CAG) stability result from the location of replication initiation relative to the repeat tract.…”

“…Current models of somatic (CTG) n •(CAG) n instability involve aberrant processing of Okazaki fragments that initiates within the repeat tract [32][33][34] . Because Okazaki processing occurs only at sites of Okazaki initiation, we thought that cis-elements that affect the frequency of Okazaki initiations in the repeat tract might affect repeat stability.…”

Concerted Action of Exonuclease and Gap-dependent Endonuclease Activities of FEN-1 Contributes to the Resolution of Triplet Repeat Sequences (CTG) - and (GAA) -derived Secondary Structures Formed during Maturation of Okazaki Fragments

“…NMR studies show that both CAG or CTG (4) and CGG (5) form stable hairpin structures, comprising a repeat unit of two GC pairs and a mismatched pair under physiological conditions. However, CAA/GTT repeats form no hairpins in vitro (6,7). All but one of the diseases, Friedreich's ataxia, occurs due to the expansion of GAA repeats in the first intron of the human frataxin gene (8).…”

“…GAA/TTC repeats that cause Friedreich ataxia have also been shown to be destabilized during DNA replication (18 -21). However, non-structure-forming GTT repeats have a 1,000-fold lower expansion rate relative to hairpin forming CNG repeats (6). This suggests that secondary structures can form in vivo and impair replication/repair enzymes.…”

“…This suggests that secondary structures can form in vivo and impair replication/repair enzymes. Studies in model organisms, such as S. cerevisiae, have shown that mutations in DNA replication enzymes, particularly Rad27 (yeast FEN-1 homolog), lead to TNR instability (6,(22)(23)(24)(25)(26)(27)(28). Rad27/FEN-1 is a multifunctional nuclease that plays a critical role in maintaining genome stability through RNA primer removal and long patch base excision repair.…”