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Results for “Flap Endonucleases”

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At least 19 recordsLinked to original sources

PubMed2001

Interaction of human AP endonuclease 1 with flap endonuclease 1 and proliferating cell nuclear antigen involved in long-patch base excision repair.

To understand the mechanism involved in the coordination of the sequential repair reactions that lead to long-patch BER, we have investigated interactions between proteins involved in this pathway. We find that human AP endonuclease 1 (APE1) physically interacts with flap endonuclease 1 (FEN1) and with proliferating cell nuclear antigen. An oligonucleotide substrate containing a reduced abasic site, which was pre-incised with APE1, was employed to reconstitute the excision step of long-patch BER with purified human DNA polymerase beta and FEN1. We demonstrate that addition of APE1 to the excision reaction mixture slightly (1.5-2-fold) stimulates the removal of the displaced flap by FEN1. These results suggest the possibility that long-patch BER is coordinated and directed by protein-protein interactions.

Carbon-Oxygen Lyases↗

PubMed2000

Inhibition of flap endonuclease 1 by flap secondary structure and relevance to repeat sequence expansion.

Recent genetic evidence indicates that null mutants of the 5'-flap endonuclease (FEN1) result in an expansion of repetitive sequences. The substrate for FEN1 is a flap formed by natural 5'-end displacement of the short intermediates of lagging strand replication. FEN1 binds the 5'-end of the flap, tracks to the point of annealing at the base of the flap, and then cleaves. Here we examine mechanisms by which foldback structures within the flap could contribute to repeat expansions. Cleavage by FEN1 was reduced with increased length of the foldback. However, even the longest foldbacks were cleaved at a low rate. Substrates containing the repetitive sequence CTG also were cleaved at a reduced rate. Bubble substrates, likely intermediates in repeat expansions, were inhibitory. Neither replication protein A nor proliferating cell nuclear antigen were able to assist in the removal of secondary structure within a flap. We propose that FEN1 cleaves natural foldbacks at a reduced rate. However, although the cleavage delay is not likely to influence the overall process of chromosomal replication, specific foldbacks could inhibit cleavage sufficiently to result in duplication of the foldback sequence.

Base Sequence↗

PubMed2002

A conserved tyrosine residue aids ternary complex formation, but not catalysis, in phage T5 flap endonuclease.

The flap endonucleases, or 5' nucleases, are involved in DNA replication and repair. They possess both 5'-3' exonucleolytic activity and the ability to cleave bifurcated, or branched DNA, in an endonucleolytic, structure-specific manner. These enzymes share a great degree of structural and sequence similarity. Conserved acidic amino acids, whose primary role appears to be chelation of essential divalent cation cofactors, lie at the base of the active site. A loop, or helical archway, is located above the active site. A conserved tyrosine residue lies at the base of the archway in phage T5 flap endonuclease. This residue is conserved in the structures of all flap endonucleases analysed to date. We mutated the tyrosine 82 codon in the cloned T5 5' nuclease to one encoding phenylalanine. Detailed analysis of the purified Y82F protein revealed only a modest (3.5-fold) decrease in binding affinity for DNA compared with wild-type in the absence of cofactor. The modified nuclease retains both structure-specific endonuclease and exonuclease activities. Kinetic analysis was performed using a newly developed single-cleavage assay based on hydrolysis of a fluorescently labelled oligonucleotide substrate. Substrate and products were resolved by denaturing HPLC. Steady-state kinetic analysis revealed that loss of the tyrosine hydroxyl function did not significantly impair k(cat). Pre-steady state analysis under single-turnover conditions also demonstrated little change in the rate of reaction compared to the wild-type protein. The pH dependence of the kinetic parameters for the Y82F enzyme-catalysed reaction was bell-shaped as for the wild-type protein. Thus, Y82 does not play a role in catalysis. However, steady-state analysis did detect a large (approximately 300-fold) defect in K(M). These results imply that this conserved tyrosine plays a key role in ternary complex formation (protein-DNA-metal ion), a prerequisite for catalysis.

Bacteriophages↗

PubMed2004

Saccharomyces cerevisiae flap endonuclease 1 uses flap equilibration to maintain triplet repeat stability.

Flap endonuclease 1 (FEN1) is a central component of Okazaki fragment maturation in eukaryotes. Genetic analysis of Saccharomyces cerevisiae FEN1 (RAD27) also reveals its important role in preventing trinucleotide repeat (TNR) expansion. In humans such expansion is associated with neurodegenerative diseases. In vitro, FEN1 can inhibit TNR expansion by employing its endonuclease activity to compete with DNA ligase I. Here we employed two yeast FEN1 nuclease mutants, rad27-G67S and rad27-G240D, to further define the mechanism by which FEN1 prevents TNR expansion. Using a yeast artificial chromosome system that can detect both TNR instability and fragility, we demonstrate that the G240D but not the G67S mutation increases both the expansion and fragility of a CTG tract in vivo. In vitro, the G240D nuclease is proficient in cleaving a fixed nonrepeat double flap; however, it exhibits severely impaired cleavage of both nonrepeat and CTG-containing equilibrating flaps. In contrast, wild-type FEN1 and the G67S mutant exhibit more efficient cleavage on an equilibrating flap than on a fixed CTG flap. The degree of TNR expansion and the amount of chromosome fragility observed in the mutant strains correlate with the severity of defective flap cleavage in vitro. We present a model to explain how flap equilibration and the unique tracking mechanism of FEN1 can collaborate to remove TNR flaps and prevent repeat expansion.

Base Sequence↗

PubMed1998

Newly discovered archaebacterial flap endonucleases show a structure-specific mechanism for DNA substrate binding and catalysis resembling human flap endonuclease-1.

Mammalian flap endonuclease-1 (FEN-1) is a structure-specific metalloenzyme that acts in processing of both the Okazaki fragments during lagging strand DNA synthesis and flap intermediates during DNA damage repair. We identified and cloned three open reading frames encoding a flap endonuclease from Archaeglobus fulgidus, Methanococcus jannaschii, and Pyrococcus furiosus, respectively. The deduced FEN-1 protein sequences share approximately 75% similarity with the human FEN-1 nuclease in the conserved nuclease domains, and extensive biochemical experiments indicate that the substrate specificities and catalytic activities of these enzymes have overall similarities with those of the human enzyme. Thus, FEN-1 enzymes and likely reaction mechanisms are conserved across the eukaryotic and archaeal kingdoms. Detailed comparative analysis, however, reveals subtle differences among these four enzymes including distinctive substrate specificity, tolerance of the archaebacterial enzymes for acidic pHs and elevated temperatures, and variations in the metal-ion dependence of substrate cleavage. Although the archaebacterial enzymes were inactive at temperatures below 30 degreesC, DNA binding occurred at temperatures as low as 4 degreesC and with or without metal ions. Thus, these archaeal enzymes may provide a means to dissect the specific binding and catalytic mechanisms of the entire FEN-1 family of structure-specific nucleases.

Acids↗

PubMedDate not supplied

Stimulation of human flap endonuclease 1 by human immunodeficiency virus type 1 integrase: possible role for flap endonuclease 1 in 5'-end processing of human immunodeficiency virus type 1 integration intermediates.

Human immunodeficiency virus type 1 (HIV-1) DNA integration intermediates consist of viral and host DNA segments separated by a 5-nucleotide gap adjacent to a 5'-AC unpaired dinucleotide. These short-flap (pre-repair) integration intermediates are structurally similar to DNA loci undergoing long-patch base excision repair in mammalian cells. The cellular proteins flap endonuclease 1 (FEN-1), proliferating cell nuclear antigen, replication factor C, DNA ligase I and DNA polymerase delta are required for the repair of this type of DNA lesion. The role of FEN-1 in the base excision repair pathway is to cleave 5'-unpaired flaps in forked structures so that DNA ligase can seal the single-stranded breaks that remain following gap repair. The rate of excision by FEN-1 of 5'-flaps from short- and long-flap oligonucleotide substrates that mimic pre- and post-repair HIV-1 integration intermediates, respectively, and the effect of HIV-1 integrase on these reactions were examined in the present study. Cleavage of 5'-flaps by FEN-1 in pre-repair HIV-1 integration intermediates was relatively inefficient and was further decreased 3-fold by HIV-1 integrase. The rate of removal of 5'-flaps by FEN-1 from post-repair HIV-1 integration intermediates containing relatively long (7-nucleotide) unpaired 5'-tails and short (1-nucleotide) gaps was increased 3-fold relative to that seen with pre-repair substrates and was further stimulated 5- to 10-fold by HIV-1 integrase. Overall, post-repair structures were cleaved 18 times more effectively in the presence of HIV-1 integrase than pre-repair structures. The site of cleavage was 1 or 2 nucleotides 3' of the branch point and was unaffected by HIV-1 integrase. Integrase alone had no detectable activity in removing 5'-flaps from either pre- or post-repair substrates.

DNA Repair↗

PubMed2002

AP endonuclease 1 coordinates flap endonuclease 1 and DNA ligase I activity in long patch base excision repair.

Base loss is common in cellular DNA, resulting from spontaneous degradation and enzymatic removal of damaged bases. Apurinic/apyrimidinic (AP) endonucleases recognize and cleave abasic (AP) sites during base excision repair (BER). APE1 (REF1, HAP1) is the predominant AP endonuclease in mammalian cells. Here we analyzed the influences of APE1 on the human BER pathway. Specifically, APE1 enhanced the enzymatic activity of both flap endonuclease1 (FEN1) and DNA ligase I. FEN1 was stimulated on all tested substrates, regardless of flap length. Interestingly, we have found that APE1 can also inhibit the activities of both enzymes on substrates with a tetrahydrofuran (THF) residue on the 5'-downstream primer of a nick, simulating a reduced abasic site. However once the THF residue was displaced at least a single nucleotide, stimulation of FEN1 activity by APE1 resumes. Stimulation of DNA ligase I required the traditional nicked substrate. Furthermore, APE1 was able to enhance overall product formation in reconstitution of BER steps involving FEN1 cleavage followed by ligation. Overall, APE1 both stimulated downstream components of BER and prevented a futile cleavage and ligation cycle, indicating a far-reaching role in BER.

Base Sequence↗

PubMed1996

Essential amino acids for substrate binding and catalysis of human flap endonuclease 1.

Human flap endonuclease 1 (FEN-1) is a member of the structure-specific endonuclease family and is involved in DNA repair. Eight restrictively conserved amino acids in FEN-1 have been converted individually to an alanine to elucidate their roles in specific DNA substrate binding and catalysis. Flap endonuclease activity of the wild type and mutant enzymes was measured by kinetic flow cytometry. Mutants D34A, D86A, and D181A lost their cleavage activity completely but retained substrate binding ability, as measured by their ability to inhibit the wild type enzyme in a competition assay. This indicates that these amino acids contribute to integrity of the enzyme active site. Loss of both binding and cleavage competency for the flap substrate by mutants E156A, G231A, and D233A suggests that these amino acids are involved in substrate binding. Mutants R103A and D179A retained wild type-like enzyme activity.

Amino Acid Sequence↗

PubMed2000

Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1.

Human flap endonuclease 1 (FEN1), an essential DNA replication protein, cleaves substrates with unannealed 5'-tails. FEN1 apparently tracks along the flap from the 5'-end to the cleavage site. Proliferating cell nuclear antigen (PCNA) stimulates FEN1 cleavage 5-50-fold. To determine whether tracking, binding, or cleavage is enhanced by PCNA, we tested a variety of flap substrates. Similar levels of PCNA stimulation occur on both a cleavage-sensitive nicked substrate and a less sensitive gapped substrate. PCNA stimulates FEN1 irrespective of the flap length. Stimulation occurs on a pseudo-Y substrate that exhibits upstream primer-independent cleavage. A pseudo-Y substrate with a sequence requiring an upstream primer for cleavage was not activated by PCNA, suggesting that PCNA does not compensate for substrate features that inhibit cleavage. A biotin.streptavidin conjugation at the 5'-end of a flap structure prevents FEN1 loading. The addition of PCNA does not restore FEN1 activity. These results indicate that PCNA does not direct FEN1 to the cleavage site from solution. Kinetic analyses reveal that PCNA can lower the K(m) for FEN1 by 11-12-fold. Overall, our results indicate that after FEN1 tracks to the cleavage site, PCNA enhances FEN1 binding stability, allowing for greater cleavage efficiency.

Base Sequence↗

PubMed2004

Triplet repeat expansion generated by DNA slippage is suppressed by human flap endonuclease 1.

Human flap endonuclease 1 (h-FEN1) mutations have dramatic effects on repeat instability. Current models for repeat expansion predict that h-FEN1 protein prevents mutations by removing 5'-flaps generated at ends of Okazaki fragments by strand displacement synthesis. The models propose that hairpin formations within flaps containing repeats enable them to escape h-FEN1 cleavage. Friedreich's ataxia is caused by expansion mutations in a d(GAA)n repeat tract. Single-stranded d(GAA)n repeat tracts, however, do not form stable hairpins until the repeat tracts are quite long. Therefore, to understand how d(GAA)n repeat expansions survive h-FEN1 activity, we determined the effects of h-FEN1 on d(GAA)n repeat expansion during replication of a d(TTC)n repeat template. Replication initiated within the repeat tract generated significant expansion that was suppressed by the addition of h-FEN1 at the start of replication. The ability of h-FEN1 to suppress expansion implies that DNA slippage generates a 5'-flap in the nascent strand independent of strand displacement synthesis by an upstream polymerase. Delaying the addition of h-FEN1 to the replication reaction abolished the ability of h-FEN1 ability to suppress d(GAA)n repeat expansion products of all sizes, including sizes unable to hairpin. Use of model substrates demonstrated that h-FEN1 cleaves d(GAA)n 5'-flaps joined to double-stranded nonrepeat sequences but not those joined to double-stranded repeat tracts. The results provide evidence that, given the opportunity, short d(GAA)n repeat expansion products rearrange from 5'-flaps to stable internal loops inside the repeat tract. Long expansion products are predicted to form hairpinned flaps and internal loops. Once formed, these DNA conformations resist h-FEN1. The biological implications of the results are discussed.

Animals↗

PubMed2005

The identification and optimization of a N-hydroxy urea series of flap endonuclease 1 inhibitors.

Flap endonuclease-1 (FEN1) is a key enzyme involved in base excision repair (BER), a primary pathway utilized by mammalian cells to repair DNA damage. Sensitization to DNA damaging agents is a potential method for the improvement of the therapeutic window of traditional chemotherapeutics. In this paper, we describe the identification and SAR of a series of low nanomolar FEN1 inhibitors. Over 1000-fold specificity was achieved against a related endonuclease, xeroderma pigmentosum G (XPG). Two compounds from this series significantly potentiate the action of methyl methanesulfonate (MMS) and temozolamide in a bladder cancer cell line (T24). To our knowledge, these are the most potent endonuclease inhibitors reported to date.

Cell Line, Tumor↗

PubMed2002

Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that cleaves substrates containing unannealed 5'-flaps during Okazaki fragment processing. Cleavage removes the flap at or near the point of annealing. The preferred substrate for archaeal FEN1 or the 5'-nuclease domains of bacterial DNA polymerases is a double-flap structure containing a 3'-tail on the upstream primer adjacent to the 5'-flap. We report that FEN1 in Saccharomyces cerevisiae (Rad27p) exhibits a similar specificity. Cleavage was most efficient when the upstream primer contained a 1-nucleotide 3'-tail as compared with the fully annealed upstream primer traditionally tested. The site of cleavage was exclusively at a position one nucleotide into the annealed region, allowing human DNA ligase I to seal all resulting nicks. In contrast, a portion of the products from traditional flap substrates is not ligated. The 3'-OH of the upstream primer is not critical for double-flap recognition, because Rad27p is tolerant of modifications. However, the positioning of the 3'-nucleotide defines the site of cleavage. We have tested substrates having complementary tails that equilibrate to many structures by branch migration. FEN1 only cleaved those containing a 1-nucleotide 3'-tail. Equilibrating substrates containing 12-ribonucleotides at the end of the 5'-flap simulates the situation in vivo. Rad27p cleaves this substrate in the expected 1-nucleotide 3'-tail configuration. Overall, these results suggest that the double-flap substrate is formed and cleaved during eukaryotic DNA replication in vivo.

Base Sequence↗

PubMed1997

Functional analysis of point mutations in human flap endonuclease-1 active site.

Human flap endonuclease-1 (hFEN-1) is highly homologous to human XPG, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1 and shares structural and functional similarity with viral exonucleases such as T4 RNase H, T5 exonuclease and prokaryotic DNA polymerase 5'nucleases. Sequence alignment of 18 structure-specific nucleases revealed two conserved nuclease domains with seven conserved carboxyl residues and one positively charged residue. In a previous report, we showed that removal of the side chain of each individual acidic residue results in complete loss of flap endonuclease activity. Here we report a detailed analysis of substrate cleavage and binding of these mutant enzymes as well as of an additional site-directed mutation of a conserved acidic residue (E160). We found that the active mutant (R103A) has substrate binding and cleavage activity indistinguishable from the wild type enzyme. Of the inactive mutants, one (D181A) has substrate binding properties comparable to the wild type, while three others (D34A, D86A and E160A) bind with lower apparent affinity (2-, 9- and 18-fold reduced, respectively). The other mutants (D158A, D179A and D233A) have no detectable binding activity. We interpret the structural implications of these findings using the crystal structures of related enzymes with the flap endonuclease activity and propose that there are two metal ions (Mg2+or Mn2+) in hFEN enzyme. These two metal coordinated active sites are distinguishable but interrelated. One metal site is directly involved in nucleophile attack to the substrate phosphodiester bonds while the other may stabilize the structure for the DNA substrate binding. These two sites may be relatively close since some of carboxyl residues can serve as ligands for both sites.

Amino Acid Sequence↗

PubMed1999

Structural changes measured by X-ray scattering from human flap endonuclease-1 complexed with Mg2+ and flap DNA substrate.

Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is essential in DNA replication and repair. FEN-1 has specific endonuclease activity for repairing nicked double-stranded DNA substrates that have the 5'-end of the nick expanded into a single-stranded tail, and it is involved in processing Okazaki fragments during DNA replication. Magnesium is a cofactor required for nuclease activity. We used small-angle x-ray scattering to obtain global structural information pertinent to nuclease activity from FEN-1, the D181A mutant, the wild-type FEN-1. 34-mer DNA flap complex, and the D181A.34-mer DNA flap complex. The D181A mutant, which has Asp-181 replaced by Ala, selectively binds to the flap structure, but has lost its cleaving activity. Asp-181 is thought to be involved in Mg2+ binding at the active site (Shen, B., Nolan, J. P., Sklar, L. A., and Park, M. S. (1996) J. Biol. Chem. 271, 9173-9176). Our data indicate that FEN-1 and the D181A mutant each have a radius of gyration of approximately 26 A, and the effect of Mg2+ on the scattering from the proteins alone is insignificant. The 34-mer DNA fragment was constructed such that it readily forms a 5'-flap structure. The formation of the flap conformation of the DNA substrate was evident by both the extrapolated Io scattering and radius of gyration and was supported by NMR spectrum and nuclease assays. In the absence of magnesium, the FEN-1.34-mer DNA flap complex has an Rg value of approximately 34 A, whereas the D181A.34-mer DNA flap complex self-associates, suggesting that a significant protein conformational change occurs by addition of the flap DNA substrate and that Asp-181 is crucial for proper binding of the protein to the DNA substrate. A time course change in the scattering profiles arising from magnesium activation of the FEN-1.34-mer DNA flap complex is consistent with the protein completely releasing the DNA substrate after cleavage.

DNA↗

PubMed2006

Flap endonuclease disengages Dna2 helicase/nuclease from Okazaki fragment flaps.

Okazaki fragments contain an initiator RNA/DNA primer that must be removed before the fragments are joined. In eukaryotes, the primer region is raised into a flap by the strand displacement activity of DNA polymerase delta. The Dna2 helicase/nuclease and then flap endonuclease 1 (FEN1) are proposed to act sequentially in flap removal. Dna2 and FEN1 both employ a tracking mechanism to enter the flap 5' end and move toward the base for cleavage. In the current model, Dna2 must enter first, but FEN1 makes the final cut at the flap base, raising the issue of how FEN1 passes the Dna2. To address this, nuclease-inactive Dna2 was incubated with a DNA flap substrate and found to bind with high affinity. FEN1 was then added, and surprisingly, there was little inhibition of FEN1 cleavage activity. FEN1 was later shown, by gel shift analysis, to remove the wild type Dna2 from the flap. RNA can be cleaved by FEN1 but not by Dna2. Pre-bound wild type Dna2 was shown to bind an RNA flap but not inhibit subsequent FEN1 cleavage. These results indicate that there is a novel interaction between the two proteins in which FEN1 disengages the Dna2 tracking mechanism. This interaction is consistent with the idea that the two proteins have evolved a special ability to cooperate in Okazaki fragment processing.

Adenosine Triphosphatases↗

PubMed2003

Functional characterization of two flap endonuclease-1 homologues in rice.

Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. Previously, we isolated and characterized a complementary DNA (cDNA) from rice (Oryza sativa) encoding a protein which shows homology with the eukaryotic flap endonuclease-1 (FEN-1). In this report, we found that rice (O. sativa L. cv. Nipponbare) possessed two FEN-1 homologues designated as OsFEN-1a and OsFEN-1b. The OsFEN-1a and OsFEN-1b genes were mapped to chromosome 5 and 3, respectively. Both genes contained 17 exons and 16 introns. Alignment of OsFEN-1a protein with OsFEN-1b protein showed a high degree of sequence similarity, particularly around the N and I domains. Northern hybridization and in situ hybridization analysis demonstrated preferential expression of OsFEN-1a and OsFEN-1b in proliferating tissues such as the shoot apical meristem or young leaves. The levels of OsFEN-1a and OsFEN-1b expression were significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium. When the growth-halted cells began to regrow following the addition of sucrose to the medium, both OsFEN-1a and OsFEN-1b were again expressed at high level. These results suggested that OsFEN-1a and OsFEN-1b are required for cell proliferation. Functional complementation assay suggested that OsFEN-1a cDNA had the ability to complement Saccharomyces cerevisiae rad27 null mutant. On the other hand, OsFEN-1b cDNA could not complement the rad27 mutant. The roles of OsFEN-1a and OsFEN-1b in plant DNA replication and repair are discussed.

Amino Acid Sequence↗

PubMed2004

Structural basis for recruitment of human flap endonuclease 1 to PCNA.

Flap endonuclease-1 (FEN1) is a key enzyme for maintaining genomic stability and replication. Proliferating cell nuclear antigen (PCNA) binds FEN1 and stimulates its endonuclease activity. The structural basis of the FEN1-PCNA interaction was revealed by the crystal structure of the complex between human FEN1 and PCNA. The main interface involves the C-terminal tail of FEN1, which forms two beta-strands connected by a short helix, the betaA-alphaA-betaB motif, participating in beta-beta and hydrophobic interactions with PCNA. These interactions are similar to those previously observed for the p21CIP1/WAF1 peptide. However, this structure involving the full-length enzyme has revealed additional interfaces that are involved in the core domain. The interactions at the interfaces maintain the enzyme in an inactive 'locked-down' orientation and might be utilized in rapid DNA-tracking by preserving the central hole of PCNA for sliding along the DNA. A hinge region present between the core domain and the C-terminal tail of FEN1 would play a role in switching the FEN1 orientation from an inactive to an active orientation.

Amino Acid Sequence↗

PubMed1999

Thermostable flap endonuclease from the archaeon, Pyrococcus horikoshii, cleaves the replication fork-like structure endo/exonucleolytically.

The flap endonuclease gene homologue from the hyperthermophilic archaeon, Pyrococcus horikoshii, was overexpressed in Escherichia coli and purified. The results of gel filtration indicated that this protein was a 41-kDa monomer. P. horikoshii flap endonuclease (phFEN) cleaves replication fork-like substrates (RF) and 5' double-strand flap structures (DF) using both flap endonuclease and 5'-3'-exonuclease activities. The mammalian flap endonuclease (mFEN) is a single-strand flap-specific endonuclease (Harrington, J. J., and Lieber, M. R. (1994) EMBO J. 13, 1235-1246), but the action patterns of phFEN appear to be quite different from those of mFEN at this point. The DF-specific flap endonuclease and 5'-exonuclease activities have not yet been reported. Therefore, this is the first report of the specific endo/exonuclease activities of phFEN. The DF-specific 5'-exonuclease activity degraded the downstream primer of 3' single-flap structure and was 15 times higher than the activities against nicked substrates without 3' flap strand. DF-specific flap endonuclease cleaved the 5' double-flap strand in DF and the lagging strand in RF at the junction portion. Because the RF appears to be the intermediate structure, due to the arrest of the replication fork, the double strand breaks after the arrests of the replication forks are probably caused by phFEN.

Amino Acid Sequence↗