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Robert A Bambara - One of the best experts on this subject based on the ideXlab platform.
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Flap Endonuclease 1
Annual review of biochemistry, 2013Co-Authors: Lata Balakrishnan, Robert A BambaraAbstract:First discovered as a structure-specific Endonuclease that evolved to cut at the base of single-stranded Flaps, Flap Endonuclease (FEN1) is now recognized as a central component of cellular DNA metabolism. Substrate specificity allows FEN1 to process intermediates of Okazaki fragment maturation, long-patch base excision repair, telomere maintenance, and stalled replication fork rescue. For Okazaki fragments, the RNA primer is displaced into a 5′ Flap and then cleaved off. FEN1 binds to the Flap base and then threads the 5′ end of the Flap through its helical arch and active site to create a configuration for cleavage. The threading requirement prevents this active nuclease from cutting the single-stranded template between Okazaki fragments. FEN1 efficiency and specificity are critical to the maintenance of genome fidelity. Overall, recent advances in our knowledge of FEN1 suggest that it was an ancient protein that has been fine-tuned over eons to coordinate many essential DNA transactions.
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Flap Endonuclease disengages dna2 helicase nuclease from okazaki fragment Flaps
Journal of Biological Chemistry, 2006Co-Authors: Jason A. Stewart, Judith L. Campbell, Robert A BambaraAbstract: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.
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Flap Endonuclease disengages DNA2 helicase/nuclease from Okazaki fragment Flaps
Journal of Biological Chemistry, 2006Co-Authors: Jason A. Stewart, Judith L. Campbell, Robert A BambaraAbstract: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.
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human bloom protein stimulates Flap Endonuclease 1 activity by resolving dna secondary structure
Journal of Biological Chemistry, 2005Co-Authors: Wensheng Wang, Robert A BambaraAbstract:Flap Endonuclease 1 (FEN1) participates in removal of RNA primers of Okazaki fragments, several DNA repair pathways, and genome stability maintenance. Defects in yeast FEN1 produce chromosomal instability, hyper-recombination, and sequence duplication. These occur because Flaps produced during replication are not promptly removed. Long-lived Flaps sustain breaks and form misaligned bubble structures that produce duplications. Flaps that can form secondary structure inhibit even wild-type FEN1 and are more likely to form bubbles. Although proliferating cell nuclear antigen stimulates FEN1, it cannot resolve secondary structures. Bloom protein (BLM) is a 3'-5' helicase, mutated in Bloom syndrome. BLM has been reported to interact with and stimulate FEN1 independent of helicase function. We found activation of the helicase by ATP did not alter BLM stimulation of cleavage of unstructured Flaps. However, BLM stimulation of FEN1 cleavage of foldback Flaps, bubbles, or triplet repeats was increased by an additional increment when ATP was added. Helicase-dependent stimulation of FEN1 cleavage was robust over a range of sizes of the single-stranded part of bubbles. However, increasing the length of the 5' annealed region of the bubble ultimately counteracted the stimulatory capacity of the BLM helicase. Moderate helicase-dependent stimulation was observed with both fixed and equilibrating CTG Flaps. Our results suggest that BLM suppresses genome instability by aiding FEN1 cleavage of structure-containing Flaps.
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the human rad9 rad1 hus1 checkpoint complex stimulates Flap Endonuclease 1
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Wensheng Wang, Patrick D. Brandt, Marie L. Rossi, Vladimir N. Podust, Ellen Fanning, Aziz Sancar, Laura A Lindseyboltz, Robert A BambaraAbstract:The toroidal damage checkpoint complex Rad9–Rad1–Hus1 (9-1-1) has been characterized as a sensor of DNA damage. Flap Endonuclease 1 (FEN1) is a structure-specific nuclease involved both in removing initiator RNA from Okazaki fragments and in DNA repair pathways. FEN1 activity is stimulated by proliferating cell nuclear antigen (PCNA), a toroidal sliding clamp that acts as a platform for DNA replication and repair complexes. We show that 9-1-1 also binds and stimulates FEN1. Stimulation is observed on a variety of Flap, nick, and gapped substrates simulating repair intermediates. Blocking 9-1-1 entry to the double strands prevents a portion of the stimulation. Like PCNA stimulation, 9-1-1 stimulation cannot circumvent the tracking mechanism by which FEN1 enters the substrate; however, 9-1-1 does not substitute for PCNA in the stimulation of DNA polymerase β. This suggests that 9-1-1 is a damage-specific activator of FEN1.
Binghui Shen - One of the best experts on this subject based on the ideXlab platform.
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Structural basis of 5' Flap recognition and protein-protein interactions of human Flap Endonuclease 1.
Nucleic acids research, 2018Co-Authors: Rongyi Shi, Li Zheng, Wanchun Han, Jiahui Cheng, Kaiying Cheng, Liangyan Wang, Bing Tian, Binghui ShenAbstract:Human Flap Endonuclease 1 (hFEN1) is a structure-specific nuclease essential for DNA replication and repair processes. hFEN1 has 5' Flap removal activity, as well as gap Endonuclease activity that is critical for restarting stalled replication forks. Here, we report the crystal structures of wild-type and mutant hFEN1 proteins in complex with DNA substrates, followed by mutagenesis studies that provide mechanistic insight into the protein-protein interactions of hFEN1. We found that in an α-helix forming the helical gateway of hFEN1 recognizes the 5' Flap prior to its threading into the active site for cleavage. We also found that the β-pin region is rigidified into a short helix in R192F hFEN1-DNA structures, suppressing its gap Endonuclease activity and cycle-dependent kinase interactions. Our findings suggest that a single mutation at the primary methylation site can alter the function of hFEN1 and provide insight into the role of the β-pin region in hFEN1 protein interactions that are essential for DNA replication and repair.
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Functional analysis of point mutations in human Flap Endonuclease-1 active site. Nucleic Acids Res. 1997; 25:3332–3338. [PubMed: 9241249
2016Co-Authors: Binghui Shen, John P. Nolan, Larry A. Sklar, Min S. ParkAbstract:Human Flap Endonuclease-1 (hFEN-1) is highly homo-logous 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 struc-tures 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
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Shade avoidance 6 encodes an Arabidopsis Flap Endonuclease required for maintenance of genome integrity and development
Nucleic acids research, 2015Co-Authors: Yijuan Zhang, Binghui Shen, Chunhong Wen, Songbai Liu, Li Zheng, Yi TaoAbstract:Flap Endonuclease-1 (FEN1) belongs to the Rad2 family of structure-specific nucleases. It is required for several DNA metabolic pathways, including DNA replication and DNA damage repair. Here, we have identified a shade avoidance mutant, sav6, which reduces the mRNA splicing efficiency of SAV6. We have demonstrated that SAV6 is an FEN1 homologue that shows double-Flap Endonuclease and gap-dependent Endonuclease activity, but lacks exonuclease activity. sav6 mutants are hypersensitive to DNA damage induced by ultraviolet (UV)-C radiation and reagents that induce double-stranded DNA breaks, but exhibit normal responses to chemicals that block DNA replication. Signalling components that respond to DNA damage are constitutively activated in sav6 mutants. These data indicate that SAV6 is required for DNA damage repair and the maintenance of genome integrity. Mutant sav6 plants also show reduced root apical meristem (RAM) size and defective quiescent centre (QC) development. The expression of SMR7, a cell cycle regulatory gene, and ERF115 and PSK5, regulators of QC division, is increased in sav6 mutants. Their constitutive induction is likely due to the elevated DNA damage responses in sav6 and may lead to defects in the development of the RAM and QC. Therefore, SAV6 assures proper root development through maintenance of genome integrity.
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Human Flap Endonuclease I is in complex with telomerase and is required for telomerase-mediated telomere maintenance.
The Journal of biological chemistry, 2008Co-Authors: Shilpa Sampathi, Binghui Shen, Amruta Bhusari, Weihang ChaiAbstract:Studies from budding yeast and ciliates have suggested that telomerase extension of telomeres requires the conventional DNA replication machinery, yet little is known about how DNA replication proteins regulate telomerase action in higher eukaryotic cells. Here we investigate the role of one of the DNA replication factors, Flap Endonuclease I (FEN1), in regulating telomerase activity in mammalian cells. FEN1 is a nuclease that plays an important role in DNA replication, repair, and recombination. We show that FEN1 is in complex with telomerase in vivo via telomeric DNA. We further demonstrate that FEN1 deficiency in mouse embryonic fibroblasts leads to an increase in telomere end-to-end fusions. In cancer cells, FEN1 deficiency induces gradual shortening of telomeres but does not alter the single-stranded G-overhangs. This is, to our knowledge, the first evidence that FEN1 and telomerase physically co-exist as a complex and that FEN1 can regulate telomerase activity at telomeres in mammalian cells.
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Overexpression and Hypomethylation of Flap Endonuclease 1 Gene in Breast and Other Cancers
Molecular cancer research : MCR, 2008Co-Authors: Purnima Singh, Ming Yang, Huifang Dai, Qin Huang, Wen Tan, Kemp H. Kernstine, Dongxin Lin, Binghui ShenAbstract:Flap Endonuclease 1 (FEN1) is a structure-specific nuclease best known for its critical roles in Okazaki fragment maturation, DNA repair, and apoptosis-induced DNA fragmentation. Functional deficiencies in FEN1, in the forms of somatic mutations and polymorphisms, have recently been shown to lead to autoimmunity, chronic inflammation, and predisposition to and progression of cancer. To explore how FEN1 contributes to cancer progression, we examined FEN1 expression using 241 matched pairs of cancer and corresponding normal tissues on a gene expression profiling array and validated differential expression by quantitative real-time PCR and immunohistochemistry. Furthermore, we defined the minimum promoter of human FEN1 and examined the methylation statuses of the 5' region of the gene in paired breast cancer tissues. We show that FEN1 is significantly up-regulated in multiple cancers and the aberrant expression of FEN1 is associated with hypomethylation of the CpG island within the FEN1 promoter in tumor cells. The overexpression and promoter hypomethylation of FEN1 may serve as biomarkers for monitoring the progression of cancers.
Vilhelm A. Bohr - One of the best experts on this subject based on the ideXlab platform.
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The human Suv3 helicase interacts with replication protein A and Flap Endonuclease 1 in the nucleus.
The Biochemical journal, 2011Co-Authors: Susanne T. Venø, Tomasz Kulikowicz, Cezar R. Pestana, Piotr P. Stepien, Tinna Stevnsner, Vilhelm A. BohrAbstract:The hSuv3 (human Suv3) helicase has been shown to be a major player in mitochondrial RNA surveillance and decay, but its physiological role might go beyond this functional niche. hSuv3 has been found to interact with BLM (Bloom's syndrome protein) and WRN (Werner's syndrome protein), members of the RecQ helicase family involved in multiple DNA metabolic processes, and in protection and stabilization of the genome. In the present study, we have addressed the possible role of hSuv3 in genome maintenance by examining its potential association with key interaction partners of the RecQ helicases. By analysis of hSuv3 co-IP (co-immunoprecipitation) complexes, we identify two new interaction partners of hSuv3: the RPA (replication protein A) and FEN1 (Flap Endonuclease 1). Utilizing an in vitro biochemical assay we find that low amounts of RPA inhibit helicase activity of hSuv3 on a forked substrate. Another single-strand-binding protein, mtSSB (mitochondrial single-strand-binding protein), fails to affect hSuv3 activity, indicating that the functional interaction is specific for hSuv3 and RPA. Further in vitro studies demonstrate that the Flap Endonuclease activity of FEN1 is stimulated by hSuv3 independently of Flap length. hSuv3 is generally thought to be a mitochondrial helicase, but the physical and functional interactions between hSuv3 and known RecQ helicase-associated proteins strengthen the hypothesis that hSuv3 may play a significant role in nuclear DNA metabolism as well.
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Human RECQL5{beta} stimulates Flap Endonuclease 1
2010Co-Authors: Elzbieta Speina, Lale Dawut, Mohammad Hedayati, Zhengming Wang, Alfred May, Sybille Schwendener, Pavel Janscak, Deborah L. Croteau, Vilhelm A. BohrAbstract:Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human Flap Endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta dramatically stimulates the rate of FEN1 cleavage of Flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.
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human recql5 beta stimulates Flap Endonuclease 1
Speina E; Dawut L; Hedayati M; Wang Z; May A; Schwendener S; Janscak P; Croteau D L; Bohr V A (2010). Human RECQL5 beta stimulates flap endonuclease 1, 2010Co-Authors: Elzbieta Speina, Lale Dawut, Mohammad Hedayati, Zhengming Wang, Alfred May, Sybille Schwendener, Pavel Janscak, Deborah L. Croteau, Vilhelm A. BohrAbstract:Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human Flap Endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta dramatically stimulates the rate of FEN1 cleavage of Flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.
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Human RECQL5β stimulates Flap Endonuclease 1
Nucleic acids research, 2010Co-Authors: Elzbieta Speina, Lale Dawut, Mohammad Hedayati, Zhengming Wang, Alfred May, Sybille Schwendener, Pavel Janscak, Deborah L. Croteau, Vilhelm A. BohrAbstract:Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human Flap Endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta dramatically stimulates the rate of FEN1 cleavage of Flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.
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Stimulation of Flap Endonuclease-1 by the Bloom's Syndrome Protein
The Journal of biological chemistry, 2003Co-Authors: Sudha Sharma, Vilhelm A. Bohr, Joshua A. Sommers, Ian D. Hickson, Robert M. BroshAbstract:Bloom's syndrome (BS) is a rare autosomal recessive genetic disorder associated with genomic instability and an elevated risk of cancer. Cellular features of BS include an accumulation of abnormal replication intermediates and increased sister chromatid exchange. Although it has been suggested that the underlying defect responsible for hyper-recombination in BS cells is a temporal delay in the maturation of DNA replication intermediates, the precise role of the BS gene product, BLM, in DNA metabolism remains elusive. We report here a novel interaction of the BLM protein with the human 5'-Flap Endonuclease/5'-3' exonuclease (FEN-1), a genome stability factor involved in Okazaki fragment processing and DNA repair. BLM protein stimulates both the endonucleolytic and exonucleolytic cleavage activity of FEN-1 and this functional interaction is independent of BLM catalytic activity. BLM and FEN-1 are associated with each other in human nuclei as shown by their reciprocal co-immunoprecipitation from HeLa nuclear extracts. The BLM-FEN-1 physical interaction is mediated through a region of the BLM C-terminal domain that shares homology with the FEN-1 interaction domain of the Werner syndrome protein, a RecQ helicase family member homologous to BLM. This study provides the first evidence for a direct interaction of BLM with a human nucleolytic enzyme. We suggest that functional interactions between RecQ helicases and Rad2 family nucleases serve to process DNA substrates that are intermediates in DNA replication and repair.
Ikuo Matsui - One of the best experts on this subject based on the ideXlab platform.
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Aromatic residues located close to the active center are essential for the catalytic reaction of Flap Endonuclease-1 from hyperthermophilic archaeon Pyrococcus horikoshii.
The Journal of biological chemistry, 2004Co-Authors: Eriko Matsui, Junko Abe, Hideshi Yokoyama, Ikuo MatsuiAbstract:Flap Endonuclease-1 (FEN-1) possessing 5'-Flap Endonuclease and 5'-->3' exonuclease activity plays important roles in DNA replication and repair. In this study, the kinetic parameters of mutants at highly conserved aromatic residues, Tyr33, Phe35, Phe79, and Phe278-Phe279, in the vicinity of the catalytic centers of FEN-1 were examined. The substitution of these aromatic residues with alanine led to a large reduction in kcat values, although these mutants retained Km values similar to that of the wild-type enzyme. Notably, the kcat of Y33A and F79A decreased 333-fold and 71-fold, respectively, compared with that of the wild-type enzyme. The aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contributed to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-Flap, and pseudo-Y substrates) for the both exo- and endo-activities, but played minor roles in recognizing the substrates with the 3' projection (the double Flap substrate and the nick substrate with the 3' projection). The replacement of Tyr33, Phe79, and Phe278-Phe279, with non-charged aromatic residues, but not with aliphatic hydrophobic residues, recovered the kcat values almost fully for the substrates without the 3' projection of the upstream strand, suggesting that the aromatic groups of Tyr33, Phe79, and Phe278-Phe279 might be involved in the catalytic reaction, probably via multiple stacking interactions with nucleotide bases. The stacking interactions of Tyr33 and Phe79 might play important roles in fixing the template strand and the downstream strand, respectively, in close proximity to the active center to achieve the productive transient state leading to the hydrolysis.
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molecular structure and novel dna binding sites located in loops of Flap Endonuclease 1 from pyrococcus horikoshii
Journal of Biological Chemistry, 2002Co-Authors: Eriko Matsui, Ikuo Matsui, Junko Abe, Krishnasastry V Musti, Kazuhiko Yamasaki, Kazuaki HarataAbstract:Abstract The crystal structure of Flap Endonuclease-1 fromPyrococcus horikoshii (phFEN-1) was determined to a resolution of 3.1 A. The active cleft of the phFEN-1 molecule is formed with one large loop and four small loops. We examined the function of the conserved residues and positively charged clusters on these loops by kinetic analysis with 45 different mutants. Arg40 and Arg42 on small loop 1, a cluster Lys193–Lys195 on small loop 2, and two sites, Arg94 and Arg118-Lys119, on the large loop were identified as binding sites. Lys87 on the large loop may play significant roles in catalytic reaction. Furthermore, we successfully elucidated the function of the four DNA binding sites that form productive ES complexes specific for each endo- or exo-type hydrolysis, probably by bending the substrates. For the endo-activity, Arg94 and Lys193–Lys195 located at the top and bottom of the molecule were key determinants. For the exo-activity, all four sites were needed, but Arg118-Lys119 was dominant. The major binding sites for both the nick substrate and double-stranded DNA might be the same.
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Thermostable Flap Endonuclease from the archaeon, Pyrococcus horikoshii, cleaves the replication fork-like structure endo/exonucleolytically.
The Journal of biological chemistry, 1999Co-Authors: Eriko Matsui, Satoko Kawasaki, Hiroyasu Ishida, Kazuhiko Ishikawa, Yoshitugu Kosugi, Kikuchi Hisashi, Kawarabayashi Yutaka, Ikuo MatsuiAbstract: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.
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thermostable Flap Endonuclease from the archaeon pyrococcus horikoshii cleaves the replication fork like structure endo exonucleolytically
Journal of Biological Chemistry, 1999Co-Authors: Eriko Matsui, Satoko Kawasaki, Hiroyasu Ishida, Kazuhiko Ishikawa, Yoshitugu Kosugi, Hisashi Kikuchi, Yutaka Kawarabayashi, Ikuo MatsuiAbstract: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.
Eriko Matsui - One of the best experts on this subject based on the ideXlab platform.
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Aromatic residues located close to the active center are essential for the catalytic reaction of Flap Endonuclease-1 from hyperthermophilic archaeon Pyrococcus horikoshii.
The Journal of biological chemistry, 2004Co-Authors: Eriko Matsui, Junko Abe, Hideshi Yokoyama, Ikuo MatsuiAbstract:Flap Endonuclease-1 (FEN-1) possessing 5'-Flap Endonuclease and 5'-->3' exonuclease activity plays important roles in DNA replication and repair. In this study, the kinetic parameters of mutants at highly conserved aromatic residues, Tyr33, Phe35, Phe79, and Phe278-Phe279, in the vicinity of the catalytic centers of FEN-1 were examined. The substitution of these aromatic residues with alanine led to a large reduction in kcat values, although these mutants retained Km values similar to that of the wild-type enzyme. Notably, the kcat of Y33A and F79A decreased 333-fold and 71-fold, respectively, compared with that of the wild-type enzyme. The aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contributed to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-Flap, and pseudo-Y substrates) for the both exo- and endo-activities, but played minor roles in recognizing the substrates with the 3' projection (the double Flap substrate and the nick substrate with the 3' projection). The replacement of Tyr33, Phe79, and Phe278-Phe279, with non-charged aromatic residues, but not with aliphatic hydrophobic residues, recovered the kcat values almost fully for the substrates without the 3' projection of the upstream strand, suggesting that the aromatic groups of Tyr33, Phe79, and Phe278-Phe279 might be involved in the catalytic reaction, probably via multiple stacking interactions with nucleotide bases. The stacking interactions of Tyr33 and Phe79 might play important roles in fixing the template strand and the downstream strand, respectively, in close proximity to the active center to achieve the productive transient state leading to the hydrolysis.
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molecular structure and novel dna binding sites located in loops of Flap Endonuclease 1 from pyrococcus horikoshii
Journal of Biological Chemistry, 2002Co-Authors: Eriko Matsui, Ikuo Matsui, Junko Abe, Krishnasastry V Musti, Kazuhiko Yamasaki, Kazuaki HarataAbstract:Abstract The crystal structure of Flap Endonuclease-1 fromPyrococcus horikoshii (phFEN-1) was determined to a resolution of 3.1 A. The active cleft of the phFEN-1 molecule is formed with one large loop and four small loops. We examined the function of the conserved residues and positively charged clusters on these loops by kinetic analysis with 45 different mutants. Arg40 and Arg42 on small loop 1, a cluster Lys193–Lys195 on small loop 2, and two sites, Arg94 and Arg118-Lys119, on the large loop were identified as binding sites. Lys87 on the large loop may play significant roles in catalytic reaction. Furthermore, we successfully elucidated the function of the four DNA binding sites that form productive ES complexes specific for each endo- or exo-type hydrolysis, probably by bending the substrates. For the endo-activity, Arg94 and Lys193–Lys195 located at the top and bottom of the molecule were key determinants. For the exo-activity, all four sites were needed, but Arg118-Lys119 was dominant. The major binding sites for both the nick substrate and double-stranded DNA might be the same.
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Thermostable Flap Endonuclease from the archaeon, Pyrococcus horikoshii, cleaves the replication fork-like structure endo/exonucleolytically.
The Journal of biological chemistry, 1999Co-Authors: Eriko Matsui, Satoko Kawasaki, Hiroyasu Ishida, Kazuhiko Ishikawa, Yoshitugu Kosugi, Kikuchi Hisashi, Kawarabayashi Yutaka, Ikuo MatsuiAbstract: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.
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thermostable Flap Endonuclease from the archaeon pyrococcus horikoshii cleaves the replication fork like structure endo exonucleolytically
Journal of Biological Chemistry, 1999Co-Authors: Eriko Matsui, Satoko Kawasaki, Hiroyasu Ishida, Kazuhiko Ishikawa, Yoshitugu Kosugi, Hisashi Kikuchi, Yutaka Kawarabayashi, Ikuo MatsuiAbstract: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.