A helical arch allowing single-stranded DNA to thread through T5 5'-exonuclease

Ceska, T.A.; Sayers, Jon R.; Stier, Günter; Suck, Dietrich

doi:10.1038/382090a0

Cited by 177 publications

(237 citation statements)

References 21 publications

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“…Comparison of the results obtained with Ps-Y and 5OVH substrates sheds light on the likely structure of the protein-DNA complex. The original DNA-binding model presented for T5 5Ј nuclease (8) is not fully compatible with the results of the quantitative binding assays presented herein. We propose a model for flap endonuclease-DNA interaction.…”

mentioning

confidence: 85%

“…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).…”

mentioning

confidence: 94%

“…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)(7)(8)(9)(10). Because none of the reported structures contained bound DNA substrates, the precise mode of nucleic acid binding is unclear.…”

mentioning

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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mentioning

confidence: 85%

“…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).…”

mentioning

confidence: 94%

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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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“…These 5Ј-nucleases are either physically linked to polymerases as in Taq polymerase and E. coli polymerase I or exist as subunits within complexes that contain discrete polymerase and nuclease activities. Examples of this latter group include the phage-encoded T5 5Ј-nucleases (4,5), archaeal (6) and mammalian flap endonuclease I (7), all of which share an ␣/␤ topology with a common central ␤-sheet and similar surrounding ␣-helices. The active site comprises a cleft formed within the central ␤-sheet; at the bottom of cleft is a set of conserved acidic residues that are essential for binding the three divalent metal ions (two Mn 2ϩ ions and one Zn 2ϩ ion) required for nuclease activity.…”

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confidence: 99%

Structure-specific DNA-induced Conformational Changes in Taq Polymerase Revealed by Small Angle Neutron Scattering

Byrnes

et al. 2004

Journal of Biological Chemistry

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The DNA polymerase I from Thermus aquaticus (Taq polymerase) performs lagging-strand DNA synthesis and DNA repair. Taq polymerase contains a polymerase domain for synthesizing a new DNA strand and a 5 -nuclease domain for cleaving RNA primers or damaged DNA strands. The extended crystal structure of Taq polymerase poses a puzzle on how this enzyme coordinates its polymerase and the nuclease activities to generate only a nick. Using contrast variation solution small angle neutron scattering, we have examined the conformational changes that occur in Taq polymerase upon binding "overlap flap" DNA, a structure-specific DNA substrate that mimics the substrate in strand replacement reactions. In solution, apoTaq polymerase has an overall expanded equilibrium conformation similar to that in the crystal structure. Upon binding to the DNA substrate, both the polymerase and the nuclease domains adopt more compact overall conformations, but these changes are not enough to bring the two active sites close enough to generate a nick. Reconstruction of the three-dimensional molecular envelope from small angle neutron scattering data shows that in the DNAbound form, the nuclease domain is lifted up relative to its position in the non-DNA-bound form so as to be in closer contact with the thumb and palm subdomains of the polymerase domain. The results suggest that a form of structure sensing is responsible for the coordination of the polymerase and nuclease activities in nick generation. However, interactions between the polymerase and the nuclease domains can assist in the transfer of the DNA substrate from one active site to the other.In addition to its well known in vitro applications in PCR, in vivo the DNA polymerase I from Thermus aquaticus (Taq polymerase) performs nucleotide replacement reactions in DNA repair and RNA primer removal in Okazaki fragment processing during lagging-strand synthesis (1). Taq polymerase also utilizes both its polymerase and 5Ј-nuclease activities in a nucleotide replacement reaction: it catalyzes the upstream addition of deoxynucleoside 5Ј-triphosphates to the 3Ј-hydroxyl terminus of an RNA primer and also cleaves the downstream, single-stranded 5Ј-nucleotide displaced by the growing upstream strand. On a DNA duplex substrate, upstream primer extension and downstream strand excision must be highly coordinated (2) to leave a ligatable nick, instead of a gap or an overhang, on a DNA duplex that is to be sealed later by a DNA ligase.The structure and functional properties of Taq polymerase are similar to those of the polymerase I of Escherichia coli (3). Taq polymerase has two distinct domains: an N-terminal domain (residues 1-289) that has single-stranded 5Ј-nucleotide cleavage activity (5Ј-nuclease domain), and a C-terminal domain (residues 306 -832) that has DNA polymerase activity (Klentaq domain). Integrated into the Klentaq domain is another domain (residues 306 -423) that is structurally analogous to the proof editing 3Ј 3 5Ј-exonuclease domain of E. coli polymerase I, but this domain has n...

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“…A first glance of the salient features of the active site of XPG was provided by crystal structures of the exonuclease domains of T4 RNase H and T5 5'-exonuclease. 19,20 These studies showed a number of conserved acidic residues that coordinate metal ions. Sequence alignments of XPG with these structures revealed that several conserved acidic residues, including Glu77, Glu791 and Asp812, are poised to be part of the active site.…”

Section: Discovery and Cloning Of Xpgmentioning

confidence: 99%

XPG: Its Products and Biological Roles

Schärer

Molecular Mechanisms of Xeroderma Pigmentosum

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Xeroderma pigmetosum patients of the complementation group G are rare. One group of XP-G patients displays a rather mild and typical XP phenotype. Mutations in these patients interfere with the function of XPG in the nucleotide excision repair, where it has a structural role in the assembly of the preincision complex and a catalytic role in making the incision 3' to the damaged site in DNA. Another set of XP-G patient is much more severely affected, displaying combined symptoms of xeroderma pigmentosum and Cockayne syndrome, referred to as XP/CS complex. Although the molecular basis leading to the XP/CS complex has not yet been fully established, current evidence suggests that these patients suffer from a mild defect in transcription in addition to a repair defect. Here, the history of how the XPG gene was discovered, the biochemical properties of the XPG protein, and the molecular defects found in XP-G patients and mouse models are reviewed.

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A helical arch allowing single-stranded DNA to thread through T5 5'-exonuclease

Cited by 177 publications

References 21 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

Structure-specific DNA-induced Conformational Changes in Taq Polymerase Revealed by Small Angle Neutron Scattering

XPG: Its Products and Biological Roles

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