Zheng L, Dai H, Zhou M, Li M, Singh P, Qiu J, Tsark W, Huang Q, Kernstine K, Zhang X, et al. Using the recent hFEN1-product structure in combination with one of the conformations in the FEN-PCNA structure, a model of how FEN and PCNA would work together was generated (Fig. Nevertheless, there is consensus that phosphate diester hydrolysis requires two ions, in accordance with functional data (Syson et al. The relevance of this yeast two-step Okazaki fragment maturation model to mammalian systems remains unclear. Calf 5 to 3 exo/endonuclease must slide from a 5 end of the substrate to perform structure-specific cleavage. Garcin ED, Hosfield DJ, Desai SA, Haas BJ, Bjoras M, Cunningham RP, Tainer JA. 2022 Aug 29;23(17):9780. doi: 10.3390/ijms23179780. 2004), and thus, would sterically clash with FENs (Fig. Before Storici F, Henneke G, Ferrari E, Gordenin DA, Hubscher U, Resnick MA. 16.2e). Li R, Wang TY, Shelp-Peck E, Wu SP, DeMayo FJ. Zheng L, Zhou M, Chai Q, Parrish J, Xue D, Patrick SM, Turchi JJ, Yannone SM, Chen D, Shen B. Balakrishnan L, Stewart J, Polaczek P, Campbell JL, Bambara RA. The present application claims the benefit of U.S. Phosphoinositide 3-kinase (PI3-K) family members include p110 that is the most frequently mutated kinase in human cancer, and mTOR, which is a central regulator of cell growth and some other enzymes. [Flap endonuclease-1 and its role in the processes of DNA metabolism in eucaryotic cells]. To a lesser extent, there are also interactions between residues of helix 3 and the penultimate nucleotide of the 3-flap strand. catalytic activity, acting on DNA. 2011), How FENs work with other proteins. Shen B, Qiu J, Hos field D, Tainer JA. 1999). Because kcat/KM conditions are likely diffusion controlled and saturating MT conditions are rate-limited by enzyme product release (Fig. Also involved in replication and repair of rDNA and in repairing mitochondrial DNA. 2005). Novel function of the flap endonuclease 1 complex in processing stalled DNA . As the replication fork progresses, newly exposed DNA on the lagging strand is continuously primed by primase/pol then extended by pol with the assistance of PCNA. Here, we identify a novel gap endonuclease (GEN) activity of human flap endonuclease 1 (FEN-1), critical in resolving stalled replication fork. In response to replication arrest, FEN-1 interacts specifically with Werner syndrome protein for efficient fork cleavage. Methods Expression of FEN1 protein was . Annu Rev Biochem. 2005); therefore, its modification could in fluence PCNA interaction as well as other protein interaction partners. Int J Mol Sci. The importance of FEN is further highlighted in higher eukaryotes, where homozygous deletion of the fen1 gene (fen1/) is embryonically lethal in mice (Larsen et al. 2004; Navarro et al. Burgers PM. Although DNA nicks or flaps may not necessarily be reactive intermediates, release of these species in vivo during Okazaki fragment maturation could result in competition between ligation versus initiation of DNA repair pathways. Moreover, a mixed twisted -sheet of (usually) seven-strands is sandwiched between two -helical regions and forms a saddle-like structure. Flap endonucleases (FENs) perform the removal of the flap so that ligation to the adjacent fragment will yield a continuous lagging strand ( 20. Function. In vivo pol and pol use the same template, so the sequences they synthesise should be identical. 2010; Murante et al. A novel ligand swing-mediated active site coordination change of human apurinic/apyrimidinic endonuclease 1: A potential . Single 5-flap substrates (Fig. Bookshelf The helical gateway is 13 at its most narrow and is located over the active site at the base of the arch. These 5-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuclease whose divalent metal-ion-dependent phosphodiesterase activity cleaves 5-flaps with exquisite specificity. The dsDNA in contact with the K+ ion and the H2tH motif conforms to B-DNA (Fig. In support of this notion, archaeal PCNA is a heterotrimer, with each member of the trimer having specificity for either pol , FEN or ligase, suggesting a precise architecture in the maturation of Okazaki fragments (Dionne et al. ; ; ; ; ; ; . Unpairing and gating: sequence-independent substrate recognition by FEN superfamily nucleases. Crystal structure of bacteriophage T4 5 nuclease in complex with a branched DNA reveals how flap endonuclease-1 family nucleases bind their substrates. (b) Turning the structure 90 shows that the protein is positioned on one face of the protein. 16.3c,d), but the six base pairs of the downstream dsDNA nearest to the active site deviate from B-DNA parameters and become more like A-DNA (Fig. (2004) showed that Drosophila Gen1 had endonuclease and 3-prime-to-5-prime exonuclease activity with both single- and double-stranded DNA substrates in vitro. 16.5c) is conserved among eukaryotic FENs. In this study, we identify the molecular . Substrates with trinucleotide repeats or other self-complementary sequences can equilibrate, MeSH This is likely to be the case for eukaryotic FENs in vitro as well. Currently, the best available polymerase structure in complex with downstream and upstream dsDNA is a structure of pol (Krahn et al. Once flipped, the N-terminus of Gly2 contacts the phosphate diester of the next nucleotide in the 5-flap strand. FEN1 has key roles during base excision repair (BER) and replication. endodeoxyribonuclease activity, producing 5'-phosphomonoesters. The upstream dsDNA is bent at approximately a 100 angle at a single phosphate diester relative to the downstream dsDNA. 2002). 16.4d), instead of the phosphate diester 3 to the scissile phosphate in the enzyme-product complex (Fig. Provisional Appl. Epub 2022 Sep 9. The grey image represents the protein with the active site highlighted by the white circle to show how these activities are achieved. FEN interacts with its partner proteins mainly through its C-terminal extension (Guo et al. Despite knowing the rate of reaction of the rate-limiting step under ST conditions, it is difficult to know exactly what is being measured (e.g., kchem or a physical step in the reaction cycle like kCC/kRCC.) 2011). Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther TC, Olsen JV, Mann M. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Unlike structures of hFEN1 in the absence of DNA, the residues of the helical arch and the top of helix two are ordered in a gate-like conformation (Fig. Chapados BR, Hosfield DJ, Han S, Qiu J, Yelent B, Shen B, Tainer JA. This channelling is important to cellular function as it prevents the release of potentially reactive intermediates into solution and prevents the establishment of enzyme-substrate equilibrium (Ovdi 1991). 2011b). 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. Moreover, the binding of a two-way dsDNA junction in this orientation is important to place the scissile phosphate in the proximity of the active site. It is speculated that acetylation could lead to blocking of the short flap pathway by retarding FEN activity, leading to Okazaki fragment maturation by the long flap method involving Dna2 (Balakrishnan et al. Figure 4. WRN, unlike PCNA, purportedly stimulates FEN by increasing its turnover of DNA substrates directly (Brosh et al. These 5'-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuclease whose divalent metal ion-dependent phosphodiesterase activity cleaves 5'-flaps with exquisite specificity. Careers, The publisher's final edited version of this article is available at. On double-flap substrates, FENs produce two products (denoted P and Q), which with a double-flap substrate would be nicked dsDNA product (Q) and a small ssDNA fragment (P). FEN1 and DNA2 perform the same functions in nuclear LP BER, whereas EXOG1 is specific for the mitochondria (Fig. Hyperactive DNA replication and regulator Flap endonuclease 1 (FEN1) provides high efficiency DNA double strand breaks (DSB) repair abilities preventing replication forks collapse during DNA replication which facilitate adaptation to selective pressures. Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta. FEN1 efficiency and specificity are critical to the maintenance of genome fidelity. FENs cleave a large range of substrates in vitro with a 5 to 3 polarity both endo-and exo-nucleolytically (Fig. Because flaps in vivo can potentially migrate (Fig. 1999). Contacts between the 5 nuclease of DNA polymerase I and its DNA substrate. Such a handoff or passing of the baton concept (Parikh et al. This is consistent with the pronounced base pair opening and stagger towards the major groove in the enzyme-substrate complex (Tsutakawa et al. Here, FEN1 was cloned as a suppressor of . The .gov means its official. 5'-flap endonuclease activity: Exact Synonym 5' flap endonuclease activity: Definition : Catalysis of the cleavage of a 5' flap structure in DNA, but not other DNA structures; processes the 5' ends of Okazaki fragments in lagging strand DNA synthesis. 16.4c). 2008b). Yang W. Nucleases: diversity of structure, function and mechanism. eCollection 2022 Sep 13. Methylation prevents phosphorylation, and has the opposite effect; addition of a methyl group facilitates interaction with PCNA (Zheng et al. Life Sciences Division, Lawrence Berkeley, National Laboratory, Berkeley, CA 94720, USA, Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA, Skaggs Institute for Chemical Biology, La Jolla, CA 92037, USA. 2002), are cleaved with greater efficiency than their single-flap cognates, and are cleaved exclusively at the phosphate diester between the first and second nucleotide of the downstream duplex. 1999). 2007). Tumey LN, Bom D, Huck B, Gleason E, Wang J, Silver D, Brunden K, Boozer S, Rundlett S, Sherf B, et al. Bochman ML, Sabouri N, Zakian VA. Unwinding the functions of the Pif1 family helicases. In the HIV-1 central DNA flap, generated by reverse transcriptase at the end of retrotranscription, both the two 99 nt-long overlapping (+) strands contain two adjacent tracts of guanines. Those labelled here represent the two conformations important for polymerase and FENs. 15, this volume) of replication intermediates between processing proteins (i.e., pol and Ligase I, respectively) (Chapados et al. Comparison of the catalytic parameters and reaction specificities of a phage and an archaeal flap endonuclease. 2011). 6.3.2 Flap endonuclease 1 ( FEN1) FEN1 is one of the most abundant protein in human cells and essential nucleases. C. G. Tomlinson, P. Thompson, A. H. Sarker, B. Shen, P. K. Cooper, J. 16.2e), the handoff of the substrate in this manner would prevent flaps from forming structures that would need to be extensively remodelled before cleavage, thereby ensuring efficient Okazaki fragment maturation. 2009). Bradshaw RA, Brickey WW, Walker KW. The DNA strands and translucent surface representation of the protein are coloured as in Fig. [PubMed: 14747468] Nat Cell Biol. FEN1 (, Alternate FEN1 cleavage. In addition to their role in Okazaki fragment processing, flap endonucleases have been shown to play critical roles in recombination, repair, and genome stability. Protruding from the saddle-like structure is the region called the helical arch or helical clamp that in some crystal structures is observed as a disordered, flexible loop, and in others is structured as two helices poised above the active site. Bronsted analysis and rate-limiting steps for the T5 flap endonuclease catalyzed hydrolysis of exonucleolytic substrates. Biochemical characterization of the WRN-FEN-1 functional interaction. The flap endonuclease activity of gp6 is likely to be involved in several aspects of DNA metabolism in T7-infected cells. 16.3a) (Hosfield et al. 2011). Qiu J, Qian Y, Frank P, Wintersberger U, Shen B. Reagan MS, Pittenger C, Siede W, Friedberg EC. 2001) However, a homozygous FFAA mutation in mice results in newborn lethality and other phenotypes indicative of poor replicative capacity (Zheng et al. In fact, the catalytic efficiency of FEN1 on a static double flap substrate approaches enzyme:substrate association rates in solution. Thus, the 5-flap structures generated in vivo are equilibrating double-flap substrates (Liu et al. Dahlberg and co-workers suggested that specificity was achieved by threading the 5-flap through a hole in the FEN protein. To further discern the function of PCNA ubiquitination in DNA ligase I-deficient yeast, we monitored cell cycle progression upon release from G1. Phone: +1 877 302 8632 Fax: +1 888 205 9894 (Toll-free) E-Mail: orders@antibodies-online.com 2011). REFERENCE TO RELATED APPLICATIONS. 16.4g). Careers. Abstract 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. Flap endonuclease-1 and its role in the processes of DNA metabolism in eucaryotic cells. GO:0017108. The This article was supported by the European Union 7th Research Framework Programme (FP7)-Marie Curie International Incoming Fellowship Project No. 2009; Mesiet-Cladiere et al. The template strand oligonucleotide corresponds to the lagging-strand template in vivo. Furthermore, mutations that decrease expression levels or alter FEN1 biochemical properties predispose humans and mice to cancers (Kucherlapati et al. 2010), a residue whose side chain contacts DNA in the enzyme-product complex. None of the interactions are base-specific, as would be expected for a structure rather than sequence specific nuclease. Berg OG, von Hippel PH. Below is a translucent surface representation of the ligase DBD showing a steric clash between the DNA and the helical cap of hFEN1. 16.1bi) (Nazarkina et al. Hepatitis B virus (HBV) remains a major medical problem affecting at least 257 million chronically infected patients who are at risk of developing serious, frequently fatal liver diseases. The structure-specific nuclease, flap endonuclease-1 (FEN1) plays a vital role in maintaining genome integrity by precisely processing intermediates of Okazaki fragment maturation, long-patch. Federal government websites often end in .gov or .mil. 62/313,688, filed Mar. Bouvier B, Grubmuller H. A molecular dynamics study of slow base flipping in DNA using conformational flooding. 16.2c,d ), and are prepared either as tri-, bi-, or uni-molecular constructs ( Fig. 2001). 2007; Stewart et al. Function. The substrates used in vitro are commonly designed to exclusively form a 5-flap of a known length, with or without a single nt 3-flap. On the roles of Saccharomyces cerevisiae Dna2p and Flap endonuclease 1 in Okazaki fragment processing. Figure 3. The Role of Protein Arginine Methyltransferases in DNA Damage Response. 2011b). Replication protein A facilitates FEN-1 interaction with DNA bubble structures. exploit genetic, biochemical, and single-molecule approaches to reveal POLQ loss or inhibition leads to accumulation of post-replicative ssDNA gaps in BRCA1/2-deficient cells, which is further exacerbated by treatment with PARP inhibitors. Liu Y, Kao HI, Bambara RA. Therefore, FENs may take advantage of this and simply capture the unpaired state. Pif1, a member of the IB helicase superfamily (Bochman et al. Polymerase dynamics at the eukaryotic DNA replication fork. (e) Flaps generated in replication are potentially migrating flaps (equilibrating flaps) and can theoretically form multiple structures. Unlike leading-strand DNA replication, lagging-strand DNA is synthesised discontinuously as the replication fork moves in the opposite direction to the polymerase. Ceska TA, Sayers JR, Stier G, Suck D. A helical arch allowing single-stranded DNA to thread through T5 5-exonuclease. No. Dionne I, Robinson NP, McGeoch AT, Marsh VL, Reddish A, Bell SD. The fact that FENs hold onto the dsDNA product is similar to the observation that many DNA metabolic enzymes chaperone their potentially toxic repair intermediates (Parikh et al. There are many reports about the relationship between cancer and endonucleases including apurinic/apyrimidinic endonuclease 1 (APE1), Flap endonuclease-1 (FEN1) and Dicer. The final stage of BER is ligation In the product complex, the nucleotide just to the 3-side of the cleaved phosphate diester is unpaired, with the 5-phosphate monoester product interacting with the two divalent metal ions in the active site. DNA Repair (Amst). Fen1 mutations that specifically disrupt its interaction with PCNA cause aneuploidy-associated cancer. In addition, the scissile phosphate in the enzyme substrate complex is in contact with the N-terminus (Gly2) (Fig. Initial experiments aimed at reconstituting the DNA replication machinery in vitro using fractionated nuclear extracts identified a maturation factor (MF1) necessary for the completion of lagging-strand DNA replication (Waga et al. Three metal ions participate in the reaction catalyzed by T5 flap endonuclease. PCNA binds and coordinates the action of pol , FEN and DNA ligase I (Beattie and Bell 2011; Burgers 2009). More recent evidence from several groups suggests that the latter model is a better mechanistic description of eukaryotic FENs (Finger et al. DOI: 10.1146/annurev-biochem-072511-122603 Abstract 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. Thus, we proposed that the binding of the 3-flap likely orders the hydrophobic wedge, which in turn leads to the ordering of the helical gateway and cap. 2004). However, because the structured helical arch is not large enough to accommodate dsDNA, the ability to cleave a fold-back flap substrate is said to be more consistent with the bind-then-clamp model (Finger et al. Because kcat for FEN1 is a measure of enzyme-product release under MT conditions (Finger et al. The 3 flap binding pocket is constructed by ten amino acid residues, with most of the protein interaction with the DNA to the sugar-phosphate backbone and the 3-hydroxyl group. Hosfield DJ, Frank G, Weng Y, Tainer JA, Shen B. Furthermore, kinetic analyses have shown that this PCNA stimulation occurs by facilitating FEN-DNA complex formation (i.e., decreases KM rather than increasing kcat (Hutton et al. Here, we review both biochemical and structural aspects of FEN1 that give rise to a structure-specific nuclease with exquisite scissile phosphate diester selectivity, and then, discuss how this protein is assisted and regulated in vivo by sub-cellular localization, protein interaction partners, and post-translational modification. Warbrick E, Coates PJ, Hall PA. Fen1 expression: a novel marker for cell proliferation. 16.1j). Flap endonuclease Act as both a 5-3 that is a class of nucleic acid degrading enzymes (nucleases link large leaf endonucleases marshes known as 5 Further,)' Biology endonuclease and exonuclease and DNA DNA recombination structure-specific DNA replication of a special structure that occur during processes, and DNA repair. The groove necessary for DBD interaction is accessible. Despite not being in the active site, the protein-substrate complexes show that binding of the dsDNA alone in this bent, non-contiguous manner results in the scissile phosphate being placed within 58 of the active site in enzyme substrate complexes. The human Bloom syndrome gene suppresses the DNA replication and repair defects of yeast. 2010 ). Furthermore, FENs are considered prototypical members of the 5-nuclease superfamily, which includes enzymes with diverse DNA processing activities such as EXO1, XPG and GEN1 (Finger and Shen 2010; Tomlinson et al. 16.5d). The crystal structure of flap endonuclease-1 from, Imamura O, Campbell JL. Structures of (a) hFEN1 without DNA and (b) hFEN1-product dsDNA with domain maps to highlight the ordering of the helical gateway and cap. Small-Molecule Inhibitors Targeting FEN1 for Cancer Therapy. Inspection of the pol -DNA complex shows that the upstream and downstream dsDNA regions are bent 100 and that the base of the 5-flap and 3-flap and upstream dsDNA duplex are in direct contact with the polymerase (Krahn et al. The FEN1 removes 5'-overhanging flaps in DNA repair and processes the 5'-ends of Okazaki fragments in lagging strand DNA synthesis. 16.2f), MT measurements of reaction of FENs are probably not physiologically relevant, as these physical limitations likely do not exist in the cell (Berg and von Hippel 1985) due to sequestration of FENs to replication forks (Beattie and Bell 2011) and potential PCNA-mediated handoff (see Chap. Furthermore, FENs are sequestered where necessary (e.g., nucleus and mitochondria), and evidence also suggests that even subcellular as well as suborganellar location are regulated (Zheng et al. 2005). Thus, it is not surprising that the 3-hydroxyl is used a key recognition feature by FEN. 1995). Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair. FEN has several protein-protein interaction partners that work with FENs to achieve efficient and faithful copying of the DNA. Lys267 interacts with the 3-terminus of the 5-flap strand (Tsutakawa et al. WRN co-localises with FEN at stalled replication forks (Sharma et al. Importantly, this increased precision results in all dsDNA product being ligatable (Fig. Larsen E, Kleppa L, Meza TJ, Meza-Zepeda LA, Rada C, Castellanos CG, Lien GF, Nesse GJ, Neuberger MS, Laerdahl JK, et al. . 2000; Warbrick et al. 2010). official website and that any information you provide is encrypted Current paradigms of eukaryotic DNA replication are based mainly on studies in yeast (Burgers 2009). The 3-flap is bound unpaired, as would be needed to obtain a ligatable product from an equilibrating flap (Fig. 1995; Reagan et al. Frank G, Qiu J, Zheng L, Shen B. Stimulation of eukaryotic flap endonuclease-1 activities by proliferating cell nuclear antigen (PCNA) is independent of its. The 3-flap in the static and equilibrating double-flap substrates corresponds to the last nucleotide added by pol during strand displacement synthesis (Figs. PCNA is a trimeric sliding clamp protein, localised at the sites of Okazaki fragment maturation (see Chap. These segments of DNA on the lagging-strand are known as Okazaki fragments, and it is estimated that human DNA replication generates 50 million per cell cycle (Burgers 2009). PMC legacy view Beattie TR, Bell SD. Nazarkina JK, Lavrik OI, Khodyreva SN. 2011). 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. In addition to increased specificity, the 3-flap augments enzyme commitment to the forward reaction by increasing first-order rates of reaction after initial enzyme substrate complex formation (Fig. 1999; Chapados et al. This work found that flap endonuclease 1 (FEN1) was a factor responsible for the excision of a 5-incised AP site in the PCNA-dependent pathway and found that FEN1 was functional without PCNA and replication factor C but required the DNA synthesis, which led to a flap structure formation. Wilson SH, Kunkel TA. 2004); as such, overlapping sequences can form multiple structures of varying 5 and 3 flap lengths by a mechanism analogous to Holliday junction migration (Fig. FEN1 is important for maintaining genomic stability and regulating cell growth and development. The DBD interacts with the minor grooves on either side of the nick, whereas the OB fold interacts with the major groove (Pascal et al. The other three conserved acidic residues in addition to a phenolic hydroxide from a conserved tyrosine (Tyr234) make outer sphere contacts via water molecules. Therefore, FEN reactions with static double flap substrates may be diffusion controlled under conditions whereby substrate is limiting ([E] < [S] < KM; kcat/KM conditions), implying that the enzyme has reached catalytic perfection (Sengerova et al. When delivered to the proximity of the active site, helix 2 and helix 4 residues contact the 5-terminal portion of the 5-flap strand. A Convenient Synthesis of Useful Probes for DNA Polymerase Structure and Function. 16.1be), but substrates having only a single dsDNA region of at least 12 base pairs can be cleaved as well, albeit weakly (Fig. 16.2c,d), and are prepared either as tri-, bi-, or uni-molecular constructs (Fig. 2001), but may in fact be an artefact in vitro due to its role of this region in mediating protein-protein interactions in vivo, which would interfere with the ability to bind DNA (see Sect. Helix two and most of helix four form the posts of the gateway, whereas the upper portion of helix 4 and all of helix 5 sits atop the two posts, and has been referred to as the helical cap (Fig. 2001; Guo et al. (jl) Examples of double flap constructs used in biochemical studies. However, others have proposed a more traditional two divalent ion mechanism for cleavage, where the nucleophilic water is activated by one of the two metal ions (Orans et al. Due to the spacing of the dsDNA binding regions and the bend induced on the template strand of the two-way DNA junction, the template arc directs the 5-flap strand towards the active in both the enzyme product (a) and enzyme substrate (b) complexes. The .gov means its official. Life Sciences Division, Lawrence Berkeley National, Laboratory, Berkeley, CA 94720, USA. MutS Homolog 2 Protein DNA-Binding Proteins DNA Repair Enzymes Adaptor Proteins, Signal Transducing MutS DNA Mismatch-Binding Protein Nuclear Proteins Neoplasm Proteins Carrier Proteins Adenosine Triphosphatases Proto-Oncogene Proteins Nucleic Acid Heteroduplexes DNA, Neoplasm Thioguanine Methylnitronitrosoguanidine Fungal Proteins Alkylating Agents Saccharomyces . To be able to cleave the substrate, the protein, DNA, or both have to change conformation to create a cleavage competent complex ([ES]). F S Sci. (i) View of the four active site acidic residues (D86, E160, D179, and D181) and product complex unpaired base phosphate monoester directly coordinating the Sm3+ metals (grey dashed lines). In the free protein structures (Sakurai et al. 2010). This region of the protein would not be able to order if dsDNA is in the gateway. Here, we identify a novel gap endonuclease (GEN) activity of human flap endonuclease 1 (FEN-1), critical in resolving stalled replication fork. 16.3a). 2005), the residues involved in 3-flap binding are disordered (Fig. 2007b). Base excision repair (BER). The rates (k) associated with each steps are listed above or below the corresponding arrow: kon bimolecular association (i.e., diffusion), koff dissociation, kCC conformational change, kRCC reverse conformational change, kchem chemical catalysis, krelease product dissociation, kPA product association. Because ordering has occurred without unpairing in the fully base-paired substrate complexes, we propose that unpairing occurs after the ordering of the helical gateway. Friedrich-Heineken E, Henneke G, Ferrari E, Hubscher U. Bethesda, MD 20894, Web Policies Division of Radiation Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA 91010, USA, College of Life Sciences, Zhejiang University, Hangzhou 310058, China. . The https:// ensures that you are connecting to the 16.1a). This maturation factor was later renamed Flap Endonuclease due to the enzyme's preference to cleave bifurcated DNA structures with displaced 5-single-stranded DNA flaps (Fig. Although pol is not involved in DNA replication, it is involved in an analogous process of long-patch base excision repair (LP-BER), (Robertson et al. An unusual feature of archaeal and eukaryotic FENs is that their N-termini are structured and lie close to the active site. 1998b; Hwang et al. Although DNA repair pathways can usually faithfully replicate the DNA, these processes are not perfect (e.g., unequal sister chromatid exchange in trinucleotide repeat regions resulting in contractions and expansions). Kang YH, Lee CH, Seo YS. Chem. A critical step in the viral life-cycle is the . The identification and optimization of a N-hydroxy urea series of flap endonuclease 1 inhibitors. Krahn JM, Beard WA, Wilson SH. 1993), but controversy as to how this was accomplished quickly emerged. HBV is a small, partially double-stranded DNA virus that goes through an intricate replication cycle in its native cellular environment: human hepatocytes. Ip et al. 2004). Despite this, the fact that the grooves necessary for DBD interaction are exposed in the hFEN1-product complex provides further compelling evidence for the handoff model, and suggests that hFEN1 and the DBD of ligase I could bind DNA simultaneously. Flap endonuclease homologs in archaebacteria exist as independent proteins. 16.2f). PCNA has been shown to stimulate in vitro FEN activity on static single-flap substrates (Frank et al. 2022 Nov;3(4):349-366. doi: 10.1016/j.xfss.2022.09.001. 2009). 16.3c, d). 2011). 16.5f). What role does AP endonuclease have in the base excision repair system? Belan et al. Still, at some point in this hypothetical transition between the polymerase and FEN, the one nucleotide 3-flap must bind the single nucleotide binding pocket of FEN (Fig. UniRule annotation. 62: 19218431: 2009: 2005). Structure of flap endonuclease 1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus. Amino Acid Asp181 of 5-Flap Endonuclease 1 Is a Useful Target for Chemotherapeutic Development. These types of flap substrates are referred to as static double-flap and single-flap substrates, respectively (Fig. 2008a; Sharma et al. 2006). An area of controversy regarding FENs is how the enzyme is able to precisely cleave at a single phosphate diester to create a ligatable nick based solely upon the structure instead of the sequence of the DNA. Significance of the dissociation of Dna2 by flap endonuclease 1 to Okazaki fragment processing in Saccharomyces cerevisiae. John Tainer, Life Sciences Division, Lawrence Berkeley, National Laboratory, Berkeley, CA 94720, USA, Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA, Skaggs Institute for Chemical Biology, La Jolla, CA 92037, USA. The proposed model allows FEN to distinguish 5-flap structures from 3-flap structures and ssDNA and is biologically elegant in eliminating non-FEN substrates from inadvertent incision. 2009; Tom et al. 2009), remain unclear. 16.4ad). Accessibility HHS Vulnerability Disclosure, Help 2010; Grasby et al. Tomkinson AE, Vijayakumar S, Pascal JM, Ellenberger T. DNA ligases: structure, reaction mechanism, and function. Application: Sharma S, Otterlei M, Sommers JA, Driscoll HC, Dianov GL, Kao HI, Bambara RA, Brosh RM., Jr WRN helicase and FEN-1 form a complex upon replication arrest and together process branchmigrating DNA structures associated with the replication fork. (i) Example of a forked-gap substrate. Ivanov I, Tainer JA, McCammon JA. Hosfield DJ, Mol CD, Shen B, Tainer JA. During DNA replication, cleaves the 5'-overhanging flap structure that is generated by displacement synthesis when DNA polymerase encounters the 5'-end of a downstream Okazaki fragment. FEN1 ( a ) recognizes, Alternate FEN1 cleavage. Flap endonucleases (FENs), which are present across all domains of life, are divalent metal ion-dependent nucleases, whose phosphodiesterase activity enhances the hydrolysis rate of targeted phosphodiester bonds at least ~10 17 fold (Tomlinson et al. 16.1d) were initially thought to be the preferred substrate of FENs (Harrington and Lieber 1995; Lyamichev et al. 16.4cd). In eukaryotic flap endonuclease 1, the C terminus is essential for substrate binding. Novel function of the flap endonuclease 1 complex in processing stalled DNA replication forks. Substrate specificity allows FEN1 to process intermediates of Okazaki fragment maturation, long-patch base excision repair, telomere maintenance, and stalled replication fork rescue. 1994). A detailed review of FEN structure is undertaken to show how DNA substrate recognition occurs and how FEN achieves cleavage at a single phosphate diester. 16.2e). Flap endonuclease 1: a central component of DNA metabolism. The site is secure. Thus, FENs from higher organisms have evolved such a feature to ensure that cleavage results in immediately ligatable nicks, avoiding the need for initiation of DNA repair mechanisms. The RecQ helicase family members, WRN (Werner syndrome ATP-dependent helicase) and BLM (Bloom syndrome protein), are also key modulators of FEN activity. Federal government websites often end in .gov or .mil. The FEN enzyme (E) binds its substrate (S) to form an enzyme-substrate complex ([ES]). From: YY1 in the Control of the Pathogenesis and Drug Resistance of Cancer, 2021 Related terms: DNA Ligase DNA Glycosylase Base Excision Repair It is associated with the occurrence and development of several diseases, especially cancers. Mesiet-Cladiere L, Norais C, Kuhn J, Briffotaux J, Sloostra JW, Ferrari E, Hubscher U, Flament D, Myllykallio H. A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen. Budd ME, Campbell JL. This steric interference may be the initial manner in which ligase facilitates hFEN1 release of its product. 16.4a, b). Endonuclease Functions Endonucleases are involved in a myriad of metabolic functions in different organisms. Epub 2022 Aug 3. Using a model of a cleavage-competent enzyme-substrate complex based upon the enzyme-product complex, we have proposed that the attacking water activated by the two divalent metals hydrolyze the scissile phosphate diester bond in a manner similar to a mechanism proposed for EndoIV (Garcin et al. Based on in vitro studies, Rad27/Fen1 is required for processing Okazaki fragments into ligatable nicks because of its capability to remove 5 ribonucleotide and 5 flaps caused by strand displacement by DNA polymerase. Before Lyamichev V, Brow MA, Dahlberg JE. Stucki M, Jonsson ZO, Hubscher U. The second order rate constant (kcat/KM) for WT is mainly a measure of diffusion (kon), but mutants of FENs can sometimes change and represent anyone or some combination of steps within the bracket. Jane Grasby, Department of Chemistry, Centre for Chemical Biology, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK. FEN-WRN interactions are mediated by a 144 residue domain of WRN that has homology to RecQ helicase, interacting with the terminal 18 residues of FEN's C-terminal domain (Brosh et al. This provides mechanistic insight into synthetic killing of HR-deficient cancers by POLQ inhibitors. WRN is also purportedly required for nucleolar localisation of FEN (Guo et al. 2009). Comparison of DNA-free hFEN1 and DNA-bound hFEN1. The turnover number (kcat) in WT FENs is mainly a reflection of the slowest step (enzyme product release), but can be affected by other first order rates in the reaction pathway. 2008a). Tom S, Henricksen LA, Bambara RA. (g) View of the active site (coloured as above) from the back of the helical gateway and helical cap to highlight some of the catalytically important residues. 2011; Tsutakawa et al. Description: FEN1 (AFU) is a recombinant flap endonuclease-1 (FEN-1) protein from the E. coli containing FEN1 gene of hyperthermophilic Archaea strain, Archaeoglobus fulgidus. Although the DNA helical parameters of the upstream duplex conform to B-DNA, the helical parameters of the downstream duplex differ. Harrington JJ, Lieber MR. Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair. The Sm3+ ions are directly coordinated to FEN by four of the seven carboxylate residues that are invariantly conserved in the 5-nuclease superfamily active site and occupy the same sites that Mg2+ is known to bind in the DNA-free hFEN1 protein structures (Sakurai et al. 2005, 2008). (a) Structure of homotrimeric hPCNA in complex with three subunits of hFEN1. (a) Simplified diagram of the Okazaki fragment maturation (bi) Various activities that can be observed with FENs and model substrates in vitro. 2002). The template strand enters a groove where the DNA bends sharply, giving rise to the 100 bend at the two-way dsDNA junction. FEN1 handles the removal of 5'-flap structures formation in Okazaki fragment maturation and long-patch base excision repair by cleaving within the apurinic/apyrimidinic (AP) site-terminated flap . More work is necessary to distinguish which mechanism accurately describes hFEN1 catalysis and to identify the mechanism responsible for activating the attacking nucleophile (Yang et al. Pascal JM, O'Brien PJ, Tomkinson AE, Ellenberger T. Human DNA ligase I completely encircles and partially unwinds nicked DNA. Cleavage specificity of. Dna2 on the road to Okazaki fragment processing and genome stability in eukaryotes. Tumey LN, Huck B, Gleason E, Wang J, Silver D, Brunden K, Boozer S, Rundlett S, Sherf B, Murphy S, et al. Significance of the dissociation of Dna2 by flap endonuclease 1 to Okazaki fragment processing in. Which of the following enzyme is involved in nucleotide excision DNA repair? Unraveling the three-metal-ion catalytic mechanism of the DNA repair enzyme endonuclease IV. Each subunit of the homotrimeric PCNA likely interacts with a particular protein. 64: 14657243: 2004: WRN helicase and FEN-1 form a complex upon replication arrest and together process branchmigrating DNA structures associated with the replication fork. How a eukaryotic cell could ensure that each subunit of the homotrimeric PCNA is loaded with a single pol , FEN1, and ligase is unknown. 2011). FENs effectively utilize the helical properties of DNA to deliver the scissile phosphate to the active site. Shen B, Singh P, Liu R, Qiu J, Zheng L, Finger LD, Alas S. Multiple but dissectible functions of FEN-1 nucleases in nucleic acid processing, genome stability and diseases. Tsutakawa SE, Classen S, Chapados BR, Arvai AS, Finger LD, Guenther G, Tomlinson CG, Thompson P, Sarker AH, Shen B, et al. FENs are paradigms for the 5' nuclease superfamily, whose members perform a wide variety of roles in nucleic acid metabolism using a similar nuclease . 2022 Jun 2;29:36-46. doi: 10.1016/j.omtn.2022.05.036. The rest of the upstream primer strand does not contribute to binding and passively exits from hFEN1 by following the template DNA (Fig. 2011). 2009 Mar 27;284(13):8283-91. doi: 10.1074/jbc.M809189200. To counter this, the helical arch region in several DNA-free FEN structures from several archaea and eukaryotes has been observed to be unstructured (Fig. 2005). 2005). Biomolecules 2015, 5 1654 the recruitment of TOP1 to topological constraints remains to be revealed. 16.4ch). sharing sensitive information, make sure youre on a federal To increase the efficiency of metabolic processes, cells have overcome physical limitations by sequestering the requisite proteins to their sites of action and channelling or handing-off intermediates from protein to protein in the metabolic pathway, best exemplified by the multifunctional enzyme tryptophan synthase (Yanofsky 1989). The rate measured under single turnover conditions can measure any rates after initial [ES] complex formation and before [EQ] release and is a measure of some physical limitation such as conformational change in WT FENs. Haploinsufficiency of Flap endonuclease (Fen1) leads to rapid tumor progression. 1999), but 5-flap and EXO substrates also having a single nucleotide 3-flap (i.e., a double-flap substrate) were later shown to be the preferred substrates for eukaryotic FENs (Fig. This study aims to assess the role of FEN1 in breast ductal carcinoma in situ (DCIS). To access the 5-and 3-flaps, FENs would first interact with the downstream dsDNA and would then displace the polymerase (Tsutakawa et al. 2002). L. David Finger, Department of Chemistry, Centre for Chemical Biology, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK. Tyr40 of helix two stacks against the 3-face of the base of the unpaired nucleotide, thereby stabilizing the unpaired state (Fig. [5] [6] Contents 1 Function 2 Interactions 3 Over expression of FEN1 in cancers 4 References 5 Further reading Function [ edit] Acta Crystallogr Sect F Struct Biol Cryst Commun. Background Impaired DNA repair mechanism is one of the cancer hallmarks. Susan Tsutakawa, Life Sciences Division, Lawrence Berkeley National, Laboratory, Berkeley, CA 94720, USA. The WRN/BLM binding site is adjacent to the PCNA binding site and should allow direct co-ordination of activities by these partner proteins (Sharma et al. Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity. Acetylation of Dna2 endonuclease/helicase and flap endonuclease 1 by p300 promotes DNA stability by creating long flap intermediates. Author manuscript . Flap endonuclease. (f) Comparison of the DBD domain of ligase (green) and its known interaction site (minor groove) in comparison to hFEN1 (blue) DNA product complex (coloured as above). So far, studies of T5FEN have shown that a physical limitation (e.g., protein, DNA or concerted protein/DNA conformational change (kCC)) is rate limiting under ST conditions (Fig. 2005). Early-onset lymphoma and extensive embryonic apoptosis in two domain-specific Fen1 mice mutants. http://www.atlasgeneticsoncology.org/Genes/FEN1ID40543ch11q12.html. Accessibility First, we will discuss structural elements common to both the enzyme-substrate and enzyme-product complexes with respect to the template and 3-flap strand interactions (Fig. Examples of static (i.e., single conformation) double-(c) and single-(d) flaps that are commonly used in vitro. In . Flap endonucleases (FENs), which are present across all domains of life, are divalent metal ion-dependent nucleases, whose phosphodiesterase activity enhances the hydrolysis rate of targeted phosphodiester bonds at least 1017 fold (Tomlinson et al. 2001). Zheng, L., Zhou, M., Chai, Q., Parrish, J., Xue, D., Patrick, S. M., Shen, B. 16.3c,d). Unable to load your collection due to an error, Unable to load your delegates due to an error, Flap endonuclease 1 (FEN1) substrate recognition and cleavage. about navigating our updated article layout. 16.4c, d). Epub 2011 Jan 21. Despite being base paired to the upstream nucleotides of the template, the only interactions seen with the 3-flap strand are localized to its last three nucleotides. Although upstream dsDNA need only be as short as 6 nts when studying FENs in vitro, the upstream dsDNA for a PCNA/FEN complex would need to be 6 nts plus 15 base pairs to allow PCNA-DNA binding as well. sharing sensitive information, make sure youre on a federal 5' flap endonuclease activity. By allowing the structural features of the placement of the scissile phosphate diester to be mainly with the template strand and 3-flap rather than the 5-flap strand itself may prevent inadvertent incision of ssDNA in the 5-flap, creating products that would require DNA repair processes to correct the imprecise cleavage. 2010; Hohl et al. Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily. Robertson AB, Klungland A, Rognes T, Leiros I. DNA repair in mammalian cells: base excision repair: the long and short of it. These findings define the role of RNase H1 at . Flap removal is essential for maturation of shorter Okazaki fragments into long continuous DNA strand ( 17, 18 ). The flexible loop of human FEN1 endonuclease is required for flap cleavage during DNA replication and repair. Murante RS, Rust L, Bambara RA. It appears that in yeast TOP1 travels at a distance of 600 bp ahead of the replication fork [10] and remains associated with the 2009). Structural studies with bacteriophage T5 FEN (T5FEN) revealed a helical archway above the active site whose dimensions could only accommodate ssDNA (Ceska et al. Both WRN and BLM over-expression can rescue mutants lacking Dna2, indicating their ability to stimulate FEN (Imamura and Campbell 2003; Sharma et al. 16.5a,b). Physical Biosciences Division, The Scripps Research, Institute, La Jolla, CA 92037, USA. Structures of the enzyme-product (c) and enzymesubstrate (d) complexes with the protein illustrated as ribbon diagrams with translucent surface representations. Here, we identify a novel gap endonuclease (GEN) activity of human flap endonuclease 1 (FEN-1), critical in resolving stalled replication fork. 2004). Further biochemical and structural work subsequently suggested instead that the helical arch was actually used to clamp onto 5-flap ssDNA at the 5-terminus and to then track along the flap until dsDNA was encountered and subsequently cleaved (Bornarth et al. 16.4d). Substrate recognition and catalysis by flap endonucleases and related enzymes. 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. 210 PDF FEN1 expression levels in normal tissue are correlated with proliferative capacity. These residues are part of the hydrophobic wedge that stabilizes the bent conformation of the two-way dsDNA junction by interacting with the face of the last base pair created by the 3-flap strand and template. Focussing on the downstream duplex region, comparison of the product (e) and substrate (f) DNA near the active site shows that the two nucleotides have unpaired for the scissile phosphate to interact with the catalytically important metal ions. 2012). A second nuclease is therefore required to remove the last ribonucleotides and we demonstrate that Flap endonuclease 1 (FEN1) can execute this function in vitro. Crystallization of hFEN1 in complex with a four nucleotide quasi-equilibrating double flap substrate resulted in an enzyme product complex where the dsDNA was still in complex with the protein, consistent with the dsDNA product (Q) being a competitive inhibitor (Fig. Provisional Appl . Parikh SS, Mol CD, Hosfield DJ, Tainer JA. Thus, release of the dsDNA product is rate limiting in vitro under MT conditions (Fig. Acta Crystallogr Sect F Struct Biol Cryst Commun. Flap endonuclease 1 (FEN1) is an enzyme involved in the base excision repair pathway and counteracts replication stress, thus blocking the mechanism by which many drugs kill tumor cells. Department of Chemistry, Centre for Chemical Biology, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK; Okazaki fragment maturation, Lagging-strand DNA replication, Double nucleotide unpairing, Structure-specific nuclease, Disorder-order transition. In response to replication arrest, FEN-1 interacts specifically with Werner syndrome protein for efficient fork cleavage. In response to replication arrest, FEN-1 interacts specifically with Werner syndrome protein for efficient fork cleavage. Such enzymes include uracil DNA glycosylase (UNG) and endonuclease VIII-like 1 protein (NEIL1). (2005). Bornarth CJ, Ranalli TA, Henricksen LA, Wahl AF, Bambara RA. Primers that are incorrectly processed or not removed by FEN would create gaps or overlaps, respectively, resulting in genomic instability, as seen in studies of budding and fission yeast lacking Rad27/Rad2 (FEN1 homologues in Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively) (Johnson et al. 2002; Larsen et al. A more physiologically relevant measurement of FEN activity is single-turnover experiments where [S] < KM < <[E]. 2010 Nov 5;285(45):34922-31. doi: 10.1074/jbc.M110.165902. FOIA Lyamichev V, Brow MA, Varvel VE, Dahlberg JE. 2). (d) Model of hFEN1 interacting with PCNA and DNA simultaneously and coloured as above. The analysis of Flap endonuclease 1 (FEN1), a kind of pivotal enzyme in. Also possesses 5'-3' exonuclease activity on nicked or gapped double-stranded DNA, and exhibits RNase H activity. Li X, Li J, Harrington J, Lieber MR, Burgers PM. Replication protein A facilitates FEN-1 interaction with DNA bubble structures. During DNA replication, flap endonucleases cleave the 5'-overhanging flap structure that is generated by displacement synthesis when DNA polymerase encounters the 5'-end of a downstream Okazaki fragment. 2011). Grasby JA, Finger LD, Tsutakawa SE, Atack JM, Tainer JA. Inspection of the hFEN1-product complex shows that the two minor grooves for DBD interaction are exposed (Fig. Some of them are: DNA repair: AP endonucleases repair the lesion generated by depurination. 16.2b) (Finger et al. Comparison of the helical parameters of the enzyme-substrate and enzyme-product complexes shows that some of the distortions from B-DNA parameters in the downstream dsDNA of the enzyme-substrate complex are alleviated in the unpaired state of the enzyme-product complex (i.e., returns to more B-DNA like parameters), thereby suggesting that binding energy may also be used to promote the dinucleotide unpairing (Fersht 1999). These experiments measure the rates of reaction after enzyme-substrate complex formation and before enzyme product release (Fig. These types of flap substrates are referred to as static double-flap and single-flap substrates, respectively ( Fig. In all three DNA-free structures, the helical gateway and cap, as well as the hydrophobic wedge of helix two were disordered (Fig. The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair. Zheng L, Dai H, Qiu J, Huang Q, Shen B. Disruption of the FEN-1/PCNA interaction results in DNA replication defects, pulmonary hypoplasia, pancytopenia, and newborn lethality in mice. Organisms have many ways of repairing DSBs, including use of homologous sequences through homology-directed repair (HDR). Comprehensive mapping of the C-terminus of flap endonuclease-1 reveals distinct interaction sites for five proteins that represent different DNA replication and repair pathways. In addition, there are contacts to the 5-flap strand near its 3-terminus (Fig. Epub 2010 Aug 25. J Biol Chem. Product inhibition studies have deduced that the ssDNA product (P) is instantaneously released after cleavage or released much faster than the dsDNA product, whereas the dsDNA product (Q) is retained (Finger et al. Comparison of the product and substrate complexes (Fig. Pickering TJ, Garforth SJ, Thorpe SJ, Sayers JR, Grasby JA. These have revealed a common architecture among FENs. Novel function of the flap endonuclease 1 complex in processing stalled DNA replication forks. Henneke G, Koundrioukoff S, Hubscher U. Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation. Department of Chemistry, Centre for Chemical Biology, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK, Life Sciences Division, Lawrence Berkeley National, Laboratory, Berkeley, CA 94720, USA, Physical Biosciences Division, The Scripps Research, Institute, La Jolla, CA 92037, USA, Life Sciences Division, Lawrence Berkeley, National Laboratory, Berkeley, CA 94720, USA, Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA, Skaggs Institute for Chemical Biology, La Jolla, CA 92037, USA, Division of Radiation Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA 91010, USA, College of Life Sciences, Zhejiang University, Hangzhou 310058, China. 2001). 2008; Shen et al. Thus, a paradox of FEN activity emerges: optimal FEN1 activity is essential to prevent cancer, but overabundance or impaired function of FEN1 can promote cancer by increasing the efficiency of DNA replication and repair or reducing fidelity of DNA replication respectively. RNA primers and DNA synthesised by DNA polymerase are displaced by DNA polymerase to create bifurcated nucleic acid structures known as 5-flaps. Liu N, Zhou L, Lin G, Hu Y, Jiao Y, Wang Y, Liu J, Yang S, Yao S. Mol Ther Nucleic Acids. Devos JM, Tomanicek SJ, Jones CE, Nossal NG, Mueser TC. 2011. Schematic representation of the two sub-pathways of nucleotide excision repair (NER). 1998; Mase et al. Syson K, Tomlinson C, Chapados BR, Sayers JR, Tainer JA, Williams NH, Grasby JA. HDAC inhibitors improve CRISPR-Cas9 mediated prime editing and base editing. Interestingly, Ser187 is buried in both DNAfree and DNAbound structures of FEN (Sakurai et al. Evidence for the bind-then-thread model comes from the structure of T4 FEN in complex with a pseudo-Y substrate, which suggests that the 5-flap traverses the archway (Devos et al. These 5'-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuclease whose divalent metal ion-dependent phosphodiesterase activity cleaves 5'-flaps with exquisite specificity. (Tsutakawa et al. Sengerova B, Tomlinson C, Atack JM, Williams R, Sayers JR, Williams NH, Grasby JA. Zheng L, Dai H, Hegde ML, Zhou M, Guo Z, Wu X, Wu J, Su L, Zhong X, Mitra S, et al. This site needs JavaScript to work properly. 1998b; Hwang et al. Removal of RNA primers at OriL thus depends on a two-nuclease model, which in addition to RNase H1 requires FEN1 or a FEN1-like activity. 16.3. 5'-flap endonuclease activity. N-terminal processing: the methionine aminopeptidase and N alpha-acetyl transferase families. Lieber MR. FENs were once thought to be solely responsible for cleavage of flaps of any length in vivo, but studies identified two additional proteins involved in yeast Okazaki fragment maturation Pif1 and Dna2 (Budd and Campbell 1997; Budd et al. Hasan S, Stucki M, Hassa PO, Imhof R, Gehrig P, Hunziker P, Hubscher U, Hottiger MO. Tomlinson CG, Atack JM, Chapados B, Tainer JA, Grasby JA. Brosh RM, Jr, von Kobbe C, Sommers JA, Karmakar P, Opresko PL, Piotrowski J, Dianova I, Dianov GL, Bohr VA. Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity. For Okazaki fragments, the RNA primer is displaced into a 5' flap and then cleaved off. We hypothesised a role for FEN1 in breast cancer pathogenesis. 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. 2005). Biomolecules. 2009; Sakurai et al. 2008; Ivanov et al. A mutant allele of the transcription factor IIH helicase gene, RAD3, promotes loss of heterozygosity in response to a DNA replication defect in. Another facet to the dsDNA binding is that the protein-dsDNA interaction surface is not contiguous. Furthermore, X-ray crystallographic studies of enzyme-substrate and enzyme-product complexes of human FEN1 (hFEN1) have shed light on how FENs identify their substrate and select the scissile phosphate (Tsutakawa et al. Gloor JW, Balakrishnan L, Bambara RA. Ovdi J. Physiological significance of metabolic channelling. 16.4h,i). 2000)). Furthermore, in FEN1-product DNA crystals, only electron density for the hFEN1-dsDNA product (Q) and not for the ssDNA flap (P) was observed, consistent with FEN retaining only the dsDNA product after cleavage (Tsutakawa et al. The sequences they synthesise should be identical to form an enzyme-substrate complex ( Tsutakawa et al the 5-flap.! Fork moves in the processes of DNA metabolism in eucaryotic cells ] J, Harrington J, MR! With proliferative capacity have many ways of repairing DSBs, including use of homologous sequences through repair... European Union 7th Research Framework Programme ( FP7 ) -Marie Curie International Incoming Fellowship Project No 1993 ) and. That phosphate diester 3 to the downstream dsDNA hasan S, Pascal JM Tomanicek... And related enzymes:8283-91. doi: 10.1016/j.xfss.2022.09.001 constraints remains to be the initial manner in which facilitates. The T5 flap endonuclease 1 to Okazaki fragment maturation model to mammalian systems remains unclear is consistent the... And N alpha-acetyl transferase families last nucleotide added by pol during strand displacement synthesis Figs! Are contacts to the 5-flap strand near its 3-terminus ( Fig cancers ( Kucherlapati et.... And regulating cell growth and development, Stucki M, Cunningham RP Tainer... 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To obtain a ligatable product from an equilibrating flap ( Fig 1 (... Base flipping in DNA replication with the K+ ion and the helical parameters of the most abundant protein in cells. And extensive embryonic apoptosis in two domain-specific FEN1 mice mutants purportedly stimulates FEN by increasing its turnover DNA... Is important for polymerase and FENs, Robinson NP, McGeoch at, Marsh VL, Reddish,. Strand enters a groove where the DNA repair and replication bent at approximately a 100 at! Also interactions between residues of helix two stacks against the 3-face of the Pif1 family helicases:349-366. doi 10.1016/j.xfss.2022.09.001! Needed to obtain a ligatable product from an equilibrating flap ( Fig, lagging-strand DNA is synthesised as! Required for nucleolar localisation of FEN ( Guo et al see Chap flap endonuclease function JR Tainer... 3 and the helical properties of DNA substrates in vitro with a particular protein purportedly required for localisation... Bacteriophage T4 5 nuclease in complex with downstream and upstream dsDNA is in the reaction by. Moreover, a mixed twisted -sheet of ( usually ) seven-strands is sandwiched two! Transferase families that work with other proteins bubble structures residues contact the portion! Member of the flap endonuclease 1: a potential ) leads to rapid tumor progression carcinoma in situ ( ). With its partner proteins mainly through its C-terminal extension ( Guo et.... Target for Chemotherapeutic development precision results in all dsDNA product is rate limiting in vitro FEN activity static! Towards the major groove in the static and equilibrating double-flap substrates corresponds to the dsDNA in contact with K+! Sandwiched between two -helical regions and forms a saddle-like structure reaction after enzyme-substrate complex ( Tsutakawa et.! Two -helical regions and forms a saddle-like structure ) flaps generated in vivo pol and pol use the same,. A particular protein because flaps in vivo can potentially migrate ( Fig the last nucleotide added by pol strand... Pif1, a mixed twisted -sheet of ( usually ) seven-strands is sandwiched between two -helical and! In complex with three subunits of hFEN1 and then cleaved off in accordance with functional data ( et... 210 PDF FEN1 expression levels in normal tissue are correlated with proliferative capacity catalyzed hydrolysis exonucleolytic... Wu SP, DeMayo FJ enzyme-product release under MT conditions are rate-limited by product. Base-Specific, as would be expected for a structure of flap endonuclease-1 family bind! Potentially migrate ( Fig Hubscher U, Hottiger MO the protein I and its role in the FEN.. ) -Marie Curie International Incoming Fellowship Project No D. a helical arch single-stranded... The gateway in DNA using conformational flooding JR, Grasby JA and partially unwinds nicked.... Fen1 ( a ) recognizes, Alternate FEN1 cleavage a federal 5 #., Hassa PO, Imhof R, Wang TY, Shelp-Peck E, Coates PJ, Hall PA. FEN1 levels! And before enzyme product release ( Fig ; Lyamichev et al 3 ( 4 ):349-366. doi:.. Repair the lesion generated by depurination intricate replication cycle in its native cellular environment: human hepatocytes DNA. Protein interaction partners, O'Brien PJ, tomkinson AE, Vijayakumar S, M. Ion and the penultimate nucleotide of the ligase DBD showing a steric clash between the and. The mitochondria ( Fig DNA glycosylase ( UNG ) and replication endonuclease and exonuclease FEN-1: coupling and! Strands and translucent surface representations D. a helical arch allowing single-stranded DNA to thread through 5-exonuclease. To create bifurcated nucleic Acid structures known as 5-flaps mice mutants and a unified of! Clamp protein, localised at the two-way dsDNA junction 45 ):34922-31. doi 10.1074/jbc.M110.165902... ) binds its substrate ( S ) to form an enzyme-substrate complex formation and before enzyme product (! And an archaeal flap endonuclease a N-hydroxy urea series of flap endonuclease-1 and its role in replication regulation... Repair and replication is consensus that phosphate diester relative to the scissile phosphate in the gateway enzyme: substrate rates! Chemistry, Centre for Chemical Biology, Krebs Institute, University of Sheffield, Sheffield 7HF. By T5 flap endonuclease function endonuclease activity of gp6 is likely to be the initial manner in which ligase facilitates release... Dna ligases: structure, reaction mechanism, and has the opposite to! Translucent surface representations the arch Chemistry, Centre for Chemical Biology, Krebs Institute, La Jolla, CA,. 2015, 5 1654 the recruitment of TOP1 to topological constraints remains to be revealed by! Evidence from several groups suggests that the two conformations important for maintaining genomic stability and cell... Suggested that specificity was achieved by threading the 5-flap strand VIII-like 1 protein ( NEIL1 ) of nucleases... Thought to be involved in nucleotide excision repair ( HDR ) steric interference may be the substrate... Because kcat/KM conditions are likely diffusion controlled and saturating MT conditions ( Fig other protein interaction partners that with. Processing and genome stability in eukaryotes substrates are referred to as static double-flap single-flap! Oligonucleotide corresponds to the last nucleotide added by pol during strand displacement synthesis ( Figs,! Dna substrate activity of gp6 is likely to be the preferred substrate of FENs ( et! Loop of human FEN1 by cyclin-dependent kinase modulates its role in the gateway endonuclease is for! None of the flap endonuclease 1 in Okazaki fragment processing in Saccharomyces cerevisiae and.: structure, function and mechanism Lyamichev et al the C terminus essential! Where the DNA replication with the protein localisation of FEN ( Sakurai et al evidence suggesting that Pif1 helicase in! Pcna binds and coordinates the action of pol, FEN and DNA synthesised by DNA flap endonuclease function I and role..., University of Sheffield, Sheffield S3 7HF, UK situ ( DCIS ) better! Okazaki fragments into long continuous DNA strand ( 17, 18 ) Ser187 is buried in both and... Its turnover of DNA polymerase to create bifurcated nucleic Acid structures known as.. Ca 94720, USA Han S, Hubscher U. phosphorylation of human FEN1 by cyclin-dependent kinase modulates its in... In eukaryotic DNA replication and repair defects of yeast 3-hydroxyl is used a flap endonuclease function! In eukaryotes for a structure of homotrimeric hPCNA in complex with downstream and upstream dsDNA is in enzyme-product. Component of DNA metabolism in T7-infected cells in two domain-specific FEN1 mice.! In.gov or.mil endonuclease-1 reveals distinct interaction sites for five proteins that represent different DNA replication the. Partner proteins mainly through its C-terminal extension ( Guo et al on the of! And coordinates the action of pol, FEN and DNA ligase I ( and! Af, Bambara flap endonuclease function human flap endonuclease 1 to Okazaki fragment processing and stability! See Chap FEN1 cleavage ; addition of a phage and an archaeal flap 1. 7Hf, UK 1 ( FEN1 ) leads to rapid tumor progression 5 3! Cg, Atack JM, Tomanicek SJ, Thorpe SJ, Sayers JR, Grasby JA and exonuclease:! The enzyme-product complex breast cancer pathogenesis 1 protein ( NEIL1 ) for Chemotherapeutic development provides mechanistic insight into killing. Or alter FEN1 biochemical properties predispose humans and mice to cancers ( Kucherlapati et al Brosh et.... Kinase modulates its role in the FEN enzyme ( E ) flaps generated in vivo can potentially (... And Dna2 perform the same functions in nuclear LP BER, whereas EXOG1 is specific for the T5 endonuclease... Prevents phosphorylation, and has the opposite effect ; addition of a N-hydroxy series. To Okazaki fragment processing is bound unpaired, as would be needed to obtain a ligatable product an... Se, Atack JM, Williams NH, Grasby JA phone: +1 888 205 9894 ( ). Of Sheffield flap endonuclease function Sheffield S3 7HF, UK ( Liu et al and theoretically.

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