A single cleavage assay for T5 5'-&gt;3' exonuclease: Determination of the catalytic parameters for wild-type and mutant proteins

Pickering, Timothy J.; Garforth, S.; Thorpe, Simon J.; Sayers, Jon R.; Grasby, Jane A.

doi:10.1093/nar/27.3.730

Cited by 23 publications

(36 citation statements)

References 25 publications

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“…This substrate undergoes a single endonucleolytic cleavage reaction to generate products of 8 and 21 nt in length (25). The catalytic parameters reported here for wild-type protein (k cat ϭ 101 min Ϫ1 , K m ϭ 70 nM) agree with those reported earlier (25).…”

Section: Resultssupporting

confidence: 87%

“…Thus, these basic residues play no role in chemical catalysis but are important for binding even for a 5OVH substrate such as HP1. The energetic penalties paid on substitution of R216 (⌬⌬G app , Table 2) or K215 (25) are consistent with the loss of either a strong hydrogen bonding interaction with an uncharged partner or a weak hydrogen bond to a charged partner (28). We previously reported k cat and K m values of 38 min Ϫ1 and 0.8 M, respectively, for K215A with ⌬⌬G app ϭ 10 kJ⅐mol Ϫ1 by using the HP1 substrate (25).…”

Section: Discussionsupporting

confidence: 57%

“…The 5OVH structure is also a good substrate for the enzyme. We previously determined a k cat of 100 min Ϫ1 by using the same hairpin 5OVH substrate (25), which compares favorably with kinetic analysis of full flap structures, e.g., k cat values of 47 min Ϫ1 for Escherichia coli DNA polymerase I and 6 min Ϫ1 for human FEN1 (16,26).…”

Section: Discussionmentioning

confidence: 99%

“…The energetic penalties paid on substitution of R216 (⌬⌬G app , Table 2) or K215 (25) are consistent with the loss of either a strong hydrogen bonding interaction with an uncharged partner or a weak hydrogen bond to a charged partner (28). We previously reported k cat and K m values of 38 min Ϫ1 and 0.8 M, respectively, for K215A with ⌬⌬G app ϭ 10 kJ⅐mol Ϫ1 by using the HP1 substrate (25). All the mutant nucleases displayed appreciable levels of activity in the spectrophotometric assay confirming that none of the residues is crucial for catalysis.…”

Section: Discussionmentioning

confidence: 99%

“…1) as described (25). HP1 concentrations were 0.005-1 M for wild type and 0.1-20 M for R216A with enzyme concentrations in the ranges of 5-30 pM for wild type and 0.12-1.8 nM for R216A.…”

Section: Methodsmentioning

confidence: 99%

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Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding

Dervan

Feng²,

Patel³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Previous structural studies on native T5 5 nuclease, a member of the flap endonuclease family of structure-specific nucleases, demonstrated that this enzyme possesses an unusual helical arch mounted on the enzyme's active site. Based on this structure, the protein's surface charge distribution, and biochemical analyses, a model of DNA binding was proposed in which single-stranded DNA threads through the archway. We investigated the kinetic and substrate-binding characteristics of wild-type and mutant nucleases in relation to the proposed model. Five basic residues R33, K215, K241, R172, and R216, are all implicated in binding branched DNA substrates. All these residues except R172 are involved in binding to duplex DNA carrying a 5 overhang. Replacement of either K215 or R216 with a neutral amino acid did not alter k cat appreciably. However, these mutant nucleases displayed significantly increased values for K d and Km. A comparison of flap endonuclease binding to pseudoY substrates and duplexes with a single-stranded 5 overhang suggests a better model for 5 nuclease-DNA binding. We propose a major revision to the binding model consistent with these biophysical data.T he flap endonucleases, or 5Ј nucleases, are structure-specific endonucleases that also possess 5Ј-3Ј exonucleolytic activity. They are involved in processing substrates with 5Ј singlestranded tails such as those that arise during nick translation and replication and in some DNA damage-repair pathways (1, 2). These enzymes bind substrates containing a single-stranded 5Ј end and a duplex region such as 5Ј overhangs (5OVHs), 5Ј flaps, and pseudoY (Ps-Y) substrates (Fig. 1;. It has been suggested that the single-stranded 5Ј tail of such substrates threads through the 5Ј nuclease (4). Several prokaryotic and archaeal flap endonuclease structures have been solved (6-10). Because none of the reported structures contained bound DNA substrates, the precise mode of nucleic acid binding is unclear. These nucleases share significant primary sequence conservation as well as extensive structural similarities (11)(12)(13)(14). A central ␤-sheet carrying many of the core metal-binding ligands is a striking feature of all 5Ј-nuclease structures reported to date. This core region contains acidic residues responsible for binding the two divalent metal ions required for nuclease activity in vitro. A series of helices and loops comprise the enzyme's core. The most variable region seems to be that comprising a helical arch in T5 5Ј nuclease, the observation of which led us to develop a model of substrate binding (ref. 8; Fig. 2). In T5 5Ј nuclease, the archway delineates a hole able to accommodate a threaded single-stranded nucleic acid. Although a similar hole is present in the Methanococcus jannaschii homologue (9), this region often appears disordered (6, 7) or as a folded loop in the structure of the Pyrococcus furiosus homologue (10). The conceptual model for flap endonuclease-DNA binding has been widely accepted (1,7,9,10,15,16). Nevertheless, this model suffers f...

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Section: Resultssupporting

confidence: 87%

Section: Discussionsupporting

confidence: 57%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…1) as described (25). HP1 concentrations were 0.005-1 M for wild type and 0.1-20 M for R216A with enzyme concentrations in the ranges of 5-30 pM for wild type and 0.12-1.8 nM for R216A.…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding

Dervan

Feng²,

Patel³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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The Wonders of Flap Endonucleases: Structure, Function, Mechanism and Regulation

Finger

Atack

Tsutakawa

et al. 2012

Subcellular Biochemistry

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Processing of Okazaki fragments to complete lagging-strand DNA synthesis requires coordination among several proteins. RNA primers and DNA synthesised by DNA polymerase α are displaced by DNA polymerase δ to create bifurcated nucleic acid structures known as 5′-flaps. 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. FENs are paradigms for the 5′ nuclease superfamily, whose members perform a wide variety of roles in nucleic acid metabolism using a similar nuclease core domain that displays common biochemical properties and structural features. 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. A proposed double nucleotide unpairing trap (DoNUT) is discussed with regards to FEN and has relevance to the wider 5′-nuclease superfamily. The homotrimeric proliferating cell nuclear antigen protein (PCNA) coordinates the actions of DNA polymerase, FEN and DNA ligase by facilitating the hand-off intermediates between each protein during Okazaki fragment maturation to maximise through-put and minimise consequences of intermediates being released into the wider cellular environment. FEN has numerous partner proteins that modulate and control its action during DNA replication and is also controlled by several post-translational modification events, all acting in concert to maintain precise and appropriate cleavage of Okazaki fragment intermediates during DNA replication.

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Characterization of a novel lytic podovirus O4 of Pseudomonas aeruginosa

et al. 2018

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Phage O4 of Pseudomonas aeruginosa was previously visualized as a short-tailed virus using a transmission electron microscope. In this work, the O4 genome was characterized to be a linear dsDNA molecule comprising 50509 bp with 76 predicted genes located in five clusters. Mass spectrometry showed that the O4 virion contains 6 putative structural proteins, 2 putative enzymes, and 7 hypothetical proteins. By analyzing a Tn5G transposon mutation library, eight genes, wbpR, wbpV, wbpO, wbpT, wbpS, wbpL, galU, and wzy, were identified and confirmed responsible for the phage-resistant phenotype; all of them are related to the synthesis of O-specific antigen (OSA) of lipopolysaccharide (LPS), indicating that OSA is the receptor for the adsorption of phage O4. Comparative genomic analysis revealed that the phage O4 genome shares little similarity to any known podovirus, indicating that phage O4 is classifiable as a novel member of the Podoviridae family.

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A single cleavage assay for T5 5'->3' exonuclease: Determination of the catalytic parameters for wild-type and mutant proteins

Cited by 23 publications

References 25 publications

Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding

Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding

The Wonders of Flap Endonucleases: Structure, Function, Mechanism and Regulation

Characterization of a novel lytic podovirus O4 of Pseudomonas aeruginosa

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