Complementary Roles for Exonuclease 1 and Flap Endonuclease 1 in Maintenance of Triplet Repeats

Abstract

Trinucleotide repeats can form stable secondary structures that promote genomic instability. To determine how such structures are resolved, we have defined biochemical activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that, consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5-flaps bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. hEXO1 did not exhibit endonuclease activity on 5-flaps bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5-flaps, but both enzymes were inhibited by G4 structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5-end, correlating with the predominance of hFEN1 endonucleolytic activity versus hEXO1 exonucleolytic activity on 5-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, respectively. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.Short tandem nucleotide repeats are widespread in mammalian genomes (1) and prone to instability because of their potential to form alternative structures during replication, transcription, or recombination. Trinucleotide repeats are of particular interest because they are associated with at least 20 human neurodegenerative disorders (2). Fragile X syndrome, the second most common cause of mental retardation, is caused by CGG repeat expansion; myotonic dystrophy by CTG repeat expansion; and Huntington chorea by CAG repeat expansions (3). All three repeats can form stable secondary structures containing stem-loops or, in the case of the CGG repeat, G4 DNA (4 -6). Formation of secondary structures can affect DNA replication, and factors that promote Okazaki fragment maturation and lagging strand synthesis have been identified as critical to maintenance of repeat stability (7-9). Secondary structure formation can also impair transcription or lead to transcription-associated instability (10 -13).FEN1 (Flap endonuclease 1) is a highly conserved RAD2 family nuclease active in DNA repair and a component of the replisome in eukaryotic cells (14). The canonical activity of FEN1 is endonucleolytic excision of a 5Ј-flap from a duplex substrate, which represents the intermediate in processing Okazaki fragments during lagging strand replication (15). FEN1 can also function as an exonuclease and gap endonuclease. FEN1 is essential in mammalian cells (16). Yeast deficient in Rad27, the FEN1 homolog, exhibits growth d...

Structures of Human Exonuclease 1 DNA Complexes Suggest a Unified Mechanism for Nuclease Family

“…Within this general framework, recognition of simple nicked (or gapped) ends, flaps, and covalently connected flaps (bubbles) or HJs can be accommodated. Accommodation of additional structures in flap regions, such as double-stranded segments, or telomeric G4 DNA (Vallur and Maizels, 2010a, b) will depend both on the distance from the nick or gap junction and potential interactions with other parts of the particular enzyme such as the highly divergent C-terminal domain. The elegant simplicity by which Nature has solved complex topological puzzles in substrate recognition and processing is remarkable.…”

RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin

“…Genetic studies have identified overlapping and distinct roles for EXO-1 and FEN-1 in replication, recombination, repair and maintenance of telomeres [21-22]. FEN-1 cleaves 5′ flaps of the branched DNA structures and possesses double-strand-specific 5′-3′ exonuclease activity [23-25].…”

DNA base excision repair: a mechanism of trinucleotide repeat expansion

“…Such inhibitory effects have been observed for CTG hairpins and CGG tetraplexes in biochemical studies [45, 49, 64], with the CTG repeat having a stronger inhibitory effect on FEN1 incision than the CGG repeat [64]. Moreover, different trinucleotide repeats can adopt distinct secondary structures that may result in different consequences for the roles of FEN1 in modulating repeat stability [64]. Other factors that could affect FEN1 are the genomic location of the CAG repeat-containing gene and its transcriptional activity.…”

“…We suggest that the data from these mouse models could indicate that the (CTG) 84 and (CGG) 130 TNR sequences are associated with stable hairpin or tetraplex structures that block FEN1 cleavage activity. Such inhibitory effects have been observed for CTG hairpins and CGG tetraplexes in biochemical studies [45, 49, 64], with the CTG repeat having a stronger inhibitory effect on FEN1 incision than the CGG repeat [64]. Moreover, different trinucleotide repeats can adopt distinct secondary structures that may result in different consequences for the roles of FEN1 in modulating repeat stability [64].…”

“…This leads to hairpin-structured CAG repeat-containing flaps along with multi-nucleotide gaps that are filled in by pol β (Figure 3b). The hairpin-containing flap inhibits “conventional” FEN1 cleavage at the base of the structured flap, and an “alternate cleavage” of a modified sugar at the 5′-end of the hairpin-structured flap can be carried out by FEN1 instead [39] [64]. This cleavage leads to production of a “ligatable nicked” intermediate (as in Figure 2b), facilitating ligation of the CAG repeat-containing hairpin into genomic DNA [39].…”