Arginine Residues 47 and 70 of Human Flap Endonuclease-1 Are Involved in DNA Substrate Interactions and Cleavage Site Determination

Qiu, Junzhuan; Bimston, David N.; Partikian, Arthur; Shen, Binghui

doi:10.1074/jbc.m111941200

Cited by 21 publications

(17 citation statements)

References 43 publications

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“…The recent structure of Archaeoglobus fulgidus FEN-1 (afFEN-1) bound to DNA identified two additional helix-loop-helix motifs that contact the upstream portion of the DNA flap substrate (29). Consistent with this report, mutation of Arg 47 and Arg 70 in human FEN-1, which are both located in these regions, affects substrate binding specificity (30). Furthermore, biochemical and mutational analysis of Pyrococcus horikoshii FEN-1 (phFEN-1) (31) revealed a total of five loop regions important for protein-DNA interactions, which include the helical arch, H3TH, and other structurally identified DNA binding regions.…”

supporting

confidence: 69%

“…Among these residues, Arg 47 is in small loop 1, which is a close neighbor to Arg 70 . Both of these residues were determined in our previous studies (30) (21)(22)(23)(24)40).…”

Section: Discussionmentioning

confidence: 99%

“…Mutations and corresponding oligonucleotide sequences for primers are listed in Table S1 for clarity as supplementary material and are available upon request. Site-directed mutagenesis, overexpression, and purification of wild type and mutant FEN-1 enzymes were carried out based on our previously published procedures (30). Mutagenesis reactions were performed using pET-28-derived plasmids harboring the wild type human FEN-1 sequence as a template so that the isolated plasmids containing a mutation could be used directly for protein expression in E. coli.…”

Section: Methodsmentioning

confidence: 99%

“…DNA Substrate Preparation and FEN-1 Nuclease Activity AssaysProtocols for DNA substrate preparation and nuclease activity assays were performed as published previously (30). Briefly, oligonucleotides as shown in the relevant figures were individually phosphorylated at the 5Ј-end.…”

Section: Methodsmentioning

confidence: 99%

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Interaction Interface of Human Flap Endonuclease-1 with Its DNA Substrates

Qiu¹,

Liu

Chapados

et al. 2004

Journal of Biological Chemistry

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Flap endonuclease-1 or FEN-1 is a structure-specific and multifunctional nuclease critical for DNA replication, repair, and recombination; however, its interaction with DNA substrates has not been fully understood. In the current study, we have defined the borders of the interaction between the FEN-1 protein and its DNA substrates and identified six clusters of conserved positively charged amino acid residues, which are in direct contact with DNA substrate. Flap endonuclease-1 (FEN-1) 1 is a critical structure-specific nuclease for ␣-segment processing during Okazaki fragment maturation and for DNA base excision repair (1-5). Deficiency of these pathways consequently results in genome instability, including significantly enhanced mutation frequency and microsatellite instability (5-11). The enzyme is also involved in preventing illegitimate crossover activities such as short repeated sequence recombination and hence enhances genome stability (12). More recently, it has been demonstrated that FEN-1 has a novel function in promoting apoptotic DNA fragmentation (13).Despite its multiple biochemical activities, protein-protein interaction partners and involvement in several DNA metabolic pathways, FEN-1 is a unique enzyme that solely recognizes abnormal DNA structures, typified by a flap DNA substrate. Initial motif analysis based on protein sequence comparison and biochemical assays identified two major conserved motifs, the N (N-terminal) and I (Intermediate) motifs, which are essential for the nuclease activities of FEN-1 proteins (4, 14). These two regions contain 7-8 conserved acidic amino acid residues that coordinate two magnesium ions and form an active center for catalysis (15). A third motif toward the C terminus is involved in the interaction between FEN-1 and proliferating cell nuclear antigen (16 -18). This interaction is required for the recruitment of FEN-1 to proliferating cell nuclear antigen at sites of Okazaki fragment processing and DNA damage repair (4,17,19). In eukaryotic cells, FEN-1 also has a C-terminal motif with multiple clusters of positively charged amino acid residues, which are important for the localization of FEN-1 into the nucleus (20).Crystal structures of FEN-1 enzymes have identified structural elements that may be important for interactions with DNA flap substrates. Structures of FEN-1 and its homologs, including three archaeal FEN-1 proteins, T4 RNase H, and T5 exonuclease (21-24) exhibit an arch element formed by a helixloop-helix motif located over the active center. This "helical arch" structure contains a number of positively charged and bulky amino acid residues on its inner side, which could be in the direct contact with the single-stranded DNA flap of DNA flap substrates (23,24). Corresponding residues in the 5Ј-nuclease of Escherichia coli DNA polymerase I have been shown to play a role in DNA binding (25). Furthermore, partial deletions of this region in Methanococcus jannaschii FEN-1 (mjFEN-1) abolish enzyme activity (24). This arch structure may allow FEN-1 to slid...

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supporting

confidence: 69%

“…Among these residues, Arg 47 is in small loop 1, which is a close neighbor to Arg 70 . Both of these residues were determined in our previous studies (30) (21)(22)(23)(24)40).…”

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Interaction Interface of Human Flap Endonuclease-1 with Its DNA Substrates

Qiu¹,

Liu

Chapados

et al. 2004

Journal of Biological Chemistry

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“…The flap endonuclease activity has been shown to require the upstream primer, which fills up the junction portion, and expanding the 3Ј of the upstream primer known as a double flap structure elevates the activity (12)(13)(14). The 5Ј33Ј exonuclease activity digested the double-stranded DNA containing the nick, gap, and 5Ј-recess-end, and the activity was elevated by expanding the 3Ј-end of the upstream primer in the nick portion (1,14).…”

mentioning

confidence: 99%

Aromatic Residues Located Close to the Active Center Are Essential for the Catalytic Reaction of Flap Endonuclease-1 from Hyperthermophilic Archaeon Pyrococcus horikoshii

Matsui

Abe

Yokoyama

et al. 2004

Journal of Biological Chemistry

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Flap endonuclease-1 (FEN-1) possessing 5-flap endonuclease and 533 exonuclease activity plays important roles in DNA replication and repair. In this study, the kinetic parameters of mutants at highly conserved aromatic residues, Tyr 33 Flap endonuclease-1 (FEN-1) 1 has important roles in DNA replication, repair, and recombination. It belongs to a family of structure-specific nucleases, which are conserved from archaea to eukaryotes and share homology with the 5Ј-nuclease domain associated with DNA polymerase I in prokaryotes and 5Ј33Ј exonuclease of bacteriophages (1-5).In DNA replication, FEN-1 removes the RNA primers during the maturation of the Okazaki fragment (6), in conjunction with Dna2 having an endonuclease activity and a helicase activity (7). For DNA repair, FEN-1 removes damaged nucleotides in long patch base excision repair (8) and is required for non-homologous end joining of double-stranded DNA breaks (9).FEN-1 possesses 5Ј-flap endonuclease and 5Ј33Ј exonuclease activities (10, 11). The flap endonuclease activity has been shown to require the upstream primer, which fills up the junction portion, and expanding the 3Ј of the upstream primer known as a double flap structure elevates the activity (12-14). The 5Ј33Ј exonuclease activity digested the double-stranded DNA containing the nick, gap, and 5Ј-recess-end, and the activity was elevated by expanding the 3Ј-end of the upstream primer in the nick portion (1,14).Three crystal structures of FEN-1 have been reported exclusively in thermophilic archaea (15-17). The molecular structure of the members of the FEN-1 family, T5 exonuclease, T4 RNase H, and the exonuclease domain of Taq polymerase were also reported (18 -20). All these structures have a conserved helical arch located above the globular domain that contains the active site, which is thought to recognize the 5Ј-end of the flap strand, tracking the length of the tail and cleaving near the junction between double-stranded and single-stranded DNA (21).Recently, DNA binding sites of FEN-1 in human, archaea, and T5 bacteriophage were identified using site-direct mutagenesis (12, 13, 17, and 22-27), and two DNA binding models were postulated based on the identified binding sites and the molecular structure (22,25). Several residues identified as DNA binding sites were located on the large loop (12, 17, 25, and 26), one of them was in contact with the bottom of the flap strand at the junction between the single-and double-stranded DNA (25). Two binding models for Pyrococcus furiosus and T5 bacteriophage FEN-1 showed that the helix-hairpin-helix region interacted with the downstream duplex DNA, and the flap strand interacted with the helical arch of FEN-1. However, the DNA binding mechanism is still unclear, because the number of DNA binding sites identified was too small to understand the substrate recognition.We previously investigated the substrate specificity of phFEN-1 in detail using 35 different substrates (1). The substrate specificity was similar to that of eukaryote FEN-1, except that phF...

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Multiple but dissectible functions of FEN‐1 nucleases in nucleic acid processing, genome stability and diseases

et al. 2005

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Flap EndoNuclease-1 (FEN-1) is a multifunctional and structure-specific nuclease involved in nucleic acid processing pathways. It plays a critical role in maintaining human genome stability through RNA primer removal, long-patch base excision repair and resolution of dinucleotide and trinucleotide repeat secondary structures. In addition to its flap endonuclease (FEN) and nick exonuclease (EXO) activities, a new gap endonuclease (GEN) activity has been characterized. This activity may be important in apoptotic DNA fragmentation and in resolving stalled DNA replication forks. The multiple functions of FEN-1 are regulated via several means, including formation of complexes with different protein partners, nuclear localization in response to cell cycle or DNA damage and post-translational modifications. Its functional deficiency is predicted to cause genetic diseases, including Huntington's disease, myotonic dystrophy and cancers. This review summarizes the knowledge gained through efforts in the past decade to define its structural elements for specific activities and possible pathological consequences of altered functions of this multirole player.

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Arginine Residues 47 and 70 of Human Flap Endonuclease-1 Are Involved in DNA Substrate Interactions and Cleavage Site Determination

Cited by 21 publications

References 43 publications

Interaction Interface of Human Flap Endonuclease-1 with Its DNA Substrates

Interaction Interface of Human Flap Endonuclease-1 with Its DNA Substrates

Aromatic Residues Located Close to the Active Center Are Essential for the Catalytic Reaction of Flap Endonuclease-1 from Hyperthermophilic Archaeon Pyrococcus horikoshii

Multiple but dissectible functions of FEN‐1 nucleases in nucleic acid processing, genome stability and diseases

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