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

Singh, Purnima; Zheng, Li; Chavez, Valerie; Qiu, Junzhuan; Shen, Binghui

doi:10.1074/jbc.m606582200

Cited by 54 publications

(53 citation statements)

References 54 publications

(85 reference statements)

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“…Protein Expression and Purification-Wild-type human FEN1 (hFEN1, accession code NP_004102) bearing a C-terminal His 6 tag was expressed from a pET28b vector in BL21(DE3) and purified as previously described (43). The protein was further purified by anion-exchange chromatography and then dialyzed against 2 ϫ 1 liter of storage buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 10% glycerol, 0.02% NaN 3 , 1 mM dithiothreitol).…”

Section: Methodsmentioning

confidence: 99%

The 3′-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

Finger¹,

Blanchard²,

Theimer

et al. 2009

Journal of Biological Chemistry

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145

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Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest especially in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that, although they share similar folds, the FEN1s of higher organisms contain a 3-extrahelical nucleotide (3-flap) binding pocket. When presented with 5-flap substrates having a 3-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well defined DNA substrates was conducted. The presence of a 3-flap on substrates reduced K m and increased multiple-and single turnover rates of endonucleolytic hydrolysis at near physiological salt concentrations. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3-flap, and the absence of a 3-flap from a 5-flap substrate was more detrimental to hFEN1 activity than removal of the 5-flap or introduction of a hairpin into the 5-flap structure. hFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5-phosphorylated product. Together the results indicate that the presence of a 3-flap is the critical feature for efficient hFEN1 substrate recognition and catalysis.In eukaryotic DNA replication and repair, various bifurcated nucleic acid structure intermediates are formed and must be processed by the appropriate nuclease. Two examples of biological processes that create bifurcated DNA intermediates are Okazaki fragment maturation (1, 2) and long patch excision repair (3). In both models, a polymerase executes strand-displacement synthesis to create a double-stranded DNA (dsDNA) 6 two-way junction from which a 5Ј-flap structure protrudes. The penultimate step of both pathways is the cleavage of this flap structure to create a nicked DNA that is then ligated. Because the bifurcated DNA structures that are formed in the aforementioned processes can theoretically occur anywhere in the genome, the nuclease associated with the cleavage of 5Ј-flap structures in eukaryotic cells, which is called flap endonuclease 1 (FEN1), must be capable of cleavage regardless of sequence. Therefore, FEN1 nucleases, which are found in all kingdoms of life (4), have evolved to recognize substrates based upon nucleic acid structure and strand polarity (5, 6).The Okazaki fragment maturation pathway of yeast has become a paradigm of eukaryotic lagging strand DNA synthesis. In the yeast model, bifurcated intermediates with large single-stranded DNA (ssDNA) 5Ј-flap structures are imprecisely cleaved by DNA2 ...

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Section: Methodsmentioning

confidence: 99%

The 3′-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

Finger¹,

Blanchard²,

Theimer

et al. 2009

Journal of Biological Chemistry

Self Cite

145

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“…The LP-BER subpathway utilizes the DNA replication machinery where FEN-1 excises the 5′-oxidized dRP-containing segment displaced as a flap during repair [82]. More recently, FEN-1 was found to have additional 5′ exonuclease and gap-specific endonuclease activities [83]. The role of FEN-1's exonuclease function in LP-BER however, is yet to be clarified.…”

Section: Two Basic Modes Of Ber: Sn-vs Lp-bermentioning

confidence: 99%

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

2008

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Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3′ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3′ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.

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“…1A). Predominance of excision activity has similarly been documented in assays of yeast Rad27 (FEN1) on structured substrates bearing CTG repeats, bubbles, and loops (28,29). hFEN1 exhibited limited flap endonuclease or 5Ј-excision activity on substrates bearing a 5Ј-flap containing a (CGG) 6 repeat (Fig.…”

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

confidence: 69%

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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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

Cited by 54 publications

References 54 publications

The 3′-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

The 3′-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

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

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