A Conserved Tyrosine Residue Aids Ternary Complex Formation, but not Catalysis, in Phage T5 Flap Endonuclease

Patel, Dipak; Tock, Mark R.; Frary, Elaine; Feng, Min; Pickering, Timothy J.; Grasby, Jane A.; Sayers, Jon R.

doi:10.1016/s0022-2836(02)00547-8

Cited by 13 publications

(22 citation statements)

References 31 publications

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“…Substrate concentrations were varied from 0.12 to 8 ϫ K m , and reactions were sampled and quenched by the addition of an equal volume of 25 mM EDTA at the appropriate time intervals. Reactions were analyzed by denaturing HPLC equipped with a fluorescence detector, and initial rates (v) were calculated as described (26 mixtures containing 0.5 or 2 mM MgCl 2 , 0.5 M HP5F, 25 mM CHES, pH 9.3, 0.1 mg/ml BSA, CaCl 2 with the appropriate amount of KCl were initiated by the addition of the appropriate amount of D201I/D204S T5FEN (final concentration 5-40 nM). All reactions were sampled and quenched as above and used to determine k obs (v/[E]) as a function of calcium ion concentration.…”

Section: Methodsmentioning

confidence: 99%

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Tomlinson

Syson

Sengerová

et al. 2011

Journal of Biological Chemistry

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Flap endonucleases (FENs) are divalent metal ion-dependent phosphodiesterases. Metallonucleases are often assigned a "two-metal ion mechanism" where both metals contact the scissile phosphate diester. The spacing of the two metal ions observed in T5FEN structures appears to preclude this mechanism. However, the overall reaction catalyzed by wild type (WT) T5FEN requires three Mg 2؉ ions, implying that a third ion is needed during catalysis, and so a two-metal ion mechanism remains possible. , essential in all life forms, are members of the 5Ј-nuclease superfamily of structure-specific phosphate diesterases that carry out essential divalent metal ion-dependent nucleic acid hydrolysis (1). FENs act upon 5Ј-bifurcated structures known as flaps formed during lagging strand DNA replication and long patch base excision repair. Hydrolysis predominantly occurs one nucleotide into the 5Ј-duplex adjoining the site of bifurcation. An unusual feature of these enzymes is their high density of active site carboxylates that coordinate essential divalent metal ion cofactors. Bacterial and bacteriophage FENs possess eight active site acidic residues, seven of which are conserved in FENs from higher organisms and also in other members of the 5Ј-nuclease superfamily (1-4). In contrast, a typical metallonuclease active site consists of three or four carboxylates (5). However, a subclass of FEN paralogues, typified by Escherichia coli ExoIX, exists in eubacteria and appear to lack three of the metal-binding carboxylates (6).Although the most common mechanism suggested for metal-dependent phosphoryl transferases involves two metal ions in contact with the scissile phosphate diester, this is not universally accepted (5, 7-10). Debate about the validity or otherwise of two-metal ion catalysis has focused on several enzymes including flap endonucleases (11-16). Crystallographic studies of substrate-free FENs have demonstrated two active site-bound metal ions (14,(17)(18)(19). Metal ion 1 (M1) occupies a similar position in all structures. However, there is some variation in the position of the site coordinating a second metal ion (M2), often observed in FEN crystal structures. The distance between the M1 and M2 sites varies, and the separations observed are generally much greater than the 4 Å required for both metals to contact the same phosphate diester. For example, in bacteriophage T5FEN, 5 the M1-M2 separation of two Mn 2ϩ ions is 8 Å (Fig. 1A) (17). However, in a substrate-free structure of hFEN1, two magnesium ions are bound with a separation of 3.4 Å in one of three proteins bound to proliferating cell nuclear antigen (Fig. 1B) , 3-(N-morpholino)propane-sulfonic acid. 5 In early literature, T5FEN was referred to as T5 5Ј-3Ј-exonuclease and T5 5Ј-nuclease. In early literature, T4FEN was referred to as referred to also as T4 RNase H.

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Section: Methodsmentioning

confidence: 99%

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Tomlinson

Syson

Sengerová

et al. 2011

Journal of Biological Chemistry

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“…This technique has been previously used for the kinetic characterization of T5FEN1 (48). Using substrates labeled with an alkyl-fluorescein moiety attached at either the 5Ј or 3Ј terminus, separation was achieved for all four hFEN1-catalyzed reactions (supplemental Fig.…”

Section: Four Different Hfen1 Substrates Prepared By Various Pairings Ofmentioning

confidence: 99%

“…To determine if this was also the case for hFEN1, single turnover (ST) experiments were performed using a quench flow apparatus under conditions whereby (23,48). Reactions were initiated by combining equal volumes of enzyme and substrate in magnesium-containing buffer and quenched with sodium hydroxide.…”

Section: Single Turnover Experiments Indicate That Product Release Ismentioning

confidence: 99%

“…The reactions were quenched after 6 min by the addition of 100 mM EDTA, 8 M urea. Reaction progress was assessed by dHPLC using a WAVE System 3500 (Trangenomics, Inc., Omaha, NB) (supplemental Table S1) with fluorescence detector and an Oligo Sep cartridge as previously described (48). The amount of product was determined by integration using Navigator TM version 1.7 (Transgenomics, Inc.) of substrate and product peaks in fluorescence intensity traces.…”

mentioning

confidence: 99%

“…Single Turnover Rapid Quench Experiments-Rapid quench experiments were conducted at 37°C using an RQF-63 device from HiTech Ltd. (Salisbury, UK) as previously described (23,48). Briefly, an 80-l aliquot of enzyme in reaction buffer (50 mM HEPES-K ϩ , pH 7.5, 0.1 mg/ml bovine serum albumin, 2.5 mM tris(3-hydroxypropyl)phosphine, 8 mM Mg(OAc) 2 , 100 mM KCl) was mixed with an equal volume of substrate in reaction buffer.…”

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

Finger¹,

Blanchard²,

Theimer

et al. 2009

Journal of Biological Chemistry

145

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

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The pH-dependence of the Escherichia coli RNase HII-catalysed Reaction Suggests that an Active Site Carboxylate Group Participates Directly in Catalysis

Bastock

Webb

Grasby

2007

Journal of Molecular Biology

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A Conserved Tyrosine Residue Aids Ternary Complex Formation, but not Catalysis, in Phage T5 Flap Endonuclease

Cited by 13 publications

References 31 publications

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

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

The pH-dependence of the Escherichia coli RNase HII-catalysed Reaction Suggests that an Active Site Carboxylate Group Participates Directly in Catalysis

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