Distinct Activities of Exonuclease 1 and Flap Endonuclease 1 at Telomeric G4 DNA

Vallur, Aarthy C.; Maizels, Nancy

doi:10.1371/journal.pone.0008908

Cited by 24 publications

(24 citation statements)

References 64 publications

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“…2, A and B). Similarly, we showed previously that telomeric sequences in a 5Ј-flap inhibit both hEXO1 exonucleolytic and 5Ј-flap endonucleolytic activities (24). Thus, like hFEN1, hEXO1 distinguishes between stem-loop and G4 DNA structures.…”

Section: Hfen1 5ј-flap Cleavage Is Impaired By Ctg or Cgg Repeats-supporting

confidence: 74%

“…If tracking does occur, then the evidence that hFEN1 predominantly excised a substrate bearing a stem-loop would suggest that this structure may impede tracking. However, hFEN1 flap cleavage was only modestly affected by a (CGG) 6 repeat within the 5Ј-flap and was unaffected by a G4 DNA structure formed by telomeric repeats at the very 5Ј-end of a flap (24). Thus, if tracking does occur, then not all 5Ј-adducts or 5Ј-structures inhibit FEN1 tracking.…”

Section: Discussionmentioning

confidence: 97%

“…Like FEN1, EXO1 is a conserved RAD2 family nuclease that possesses both 5Ј-flap endonuclease and exonucleolytic activities and has important functions in replication, repair, and recombination (14,19,20). Genetic analysis suggests that the activities of EXO1 and FEN1 might be partially redundant, as overexpression of Exo1 is able to rescue the mutator phenotype of yeast rad27 (21), whereas rad27⌬ is synthetic lethal when combined with exo1⌬ in yeast (22).We showed recently that purified human (h) 2 EXO1 acts on flap substrates and transcribed substrates containing G4 DNA (23,24). This prompted us to compare the activities of purified hEXO1 and hFEN1 on substrates recapitulating replication intermediates bearing CTG or CGG repeats.…”

mentioning

confidence: 99%

“…We showed recently that purified human (h) 2 EXO1 acts on flap substrates and transcribed substrates containing G4 DNA (23,24). This prompted us to compare the activities of purified hEXO1 and hFEN1 on substrates recapitulating replication intermediates bearing CTG or CGG repeats.…”

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confidence: 99%

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

Vallur

Maizels

2010

Journal of Biological Chemistry

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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...

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Section: Hfen1 5ј-flap Cleavage Is Impaired By Ctg or Cgg Repeats-supporting

confidence: 74%

Section: Discussionmentioning

confidence: 97%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Vallur

Maizels

2010

Journal of Biological Chemistry

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“…Since then EXO1 has been implicated in a multitude of eukaryotic DNA metabolic pathways and in maintaining genomic integrity. It is involved in DNA mismatch repair (MMR) by hydrolyzing DNA mismatches (2)(3)(4), in DNA double-strand break repair (DSBR) through DNA end resection (5-7), in B-cell development through the generation of antibody diversity (8), and in telomere maintenance by promotion of telomeric recombination (9). Biochemical analysis had shown that the N-terminal half of EXO1 possesses 5′-3′ exonuclease and 5′ flap-endonuclease activities (10).…”

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confidence: 99%

MammalianExo1encodes both structural and catalytic functions that play distinct roles in essential biological processes

Schaetzlein¹,

Chahwan²,

Avdievich³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Mammalian Exonuclease 1 (EXO1) is an evolutionarily conserved, multifunctional exonuclease involved in DNA damage repair, replication, immunoglobulin diversity, meiosis, and telomere maintenance. It has been assumed that EXO1 participates in these processes primarily through its exonuclease activity, but recent studies also suggest that EXO1 has a structural function in the assembly of higher-order protein complexes. To dissect the enzymatic and nonenzymatic roles of EXO1 in the different biological processes in vivo, we generated an EXO1-E109K knockin (Exo1 EK ) mouse expressing a stable exonuclease-deficient protein and, for comparison, a fully EXO1-deficient (Exo1 null ) mouse. In contrast to Exo1 null/null mice, Exo1 EK/EK mice retained mismatch repair activity and displayed normal class switch recombination and meiosis. However, both Exo1-mutant lines showed defects in DNA damage response including DNA double-strand break repair (DSBR) through DNA end resection, chromosomal stability, and tumor suppression, indicating that the enzymatic function is required for those processes. On a transformation-related protein 53 (Trp53)-null background, the DSBR defect caused by the E109K mutation altered the tumor spectrum but did not affect the overall survival as compared with p53-Exo1 null mice, whose defects in both DSBR and mismatch repair also compromised survival. The separation of these functions demonstrates the differential requirement for the structural function and nuclease activity of mammalian EXO1 in distinct DNA repair processes and tumorigenesis in vivo.somatic hypermuation | scaffold function | ssDNA E xonuclease 1 (EXO1) belongs to the XPG/Rad2 family of metallonucleases and was first described as a 5′-3′ exonuclease associated with meiosis in Schizosaccharomyces pombe (1). Since then EXO1 has been implicated in a multitude of eukaryotic DNA metabolic pathways and in maintaining genomic integrity. It is involved in DNA mismatch repair (MMR) by hydrolyzing DNA mismatches (2-4), in DNA double-strand break repair (DSBR) through DNA end resection (5-7), in B-cell development through the generation of antibody diversity (8), and in telomere maintenance by promotion of telomeric recombination (9). Biochemical analysis had shown that the N-terminal half of EXO1 possesses 5′-3′ exonuclease and 5′ flap-endonuclease activities (10). However, these apparently distinct functions now are thought to be mechanistically unified (11).MMR is essential for maintaining the integrity of eukaryotic genomes by removing misincorporated nucleotides that result from erroneous replication. During MMR, the repair of distinct types of mismatches is initiated by two partially redundant MutS homolog (MSH) complexes: the MSH2-MSH6 (MutSα) heterodimer, that recognizes and binds to single-base mispairs and single-base insertion/deletions, and the MSH2-MSH3 (MutSβ) complex that primarily interacts with single-base and larger insertions/deletions. Subsequent to mismatch recognition by the MSH complexes, a MutL homolog (MLH) complex con...

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

Orans

McSweeney

Iyer

et al. 2011

Cell

140

285

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Summary Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5′ structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. hExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5′ ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exo-nucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant solution to a complex geometrical puzzle of substrate recognition and processing.

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Distinct Activities of Exonuclease 1 and Flap Endonuclease 1 at Telomeric G4 DNA

Cited by 24 publications

References 64 publications

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

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

MammalianExo1encodes both structural and catalytic functions that play distinct roles in essential biological processes

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

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