Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' exonucleolytic active site of the Klenow fragment

Brautigam, Chad A.; Aschheim, Kathryn; Steitz, Thomas A.

doi:10.1016/s1074-5521(00)80009-5

Cited by 37 publications

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“…The presence of other metals, such as zinc, at site A has been described, although with magnesium at site B, for the Klenow exonuclease fragment of E. coli DNA polymerase III (Brautigam and Steitz 1998;Brautigam et al 1999b), and has been related to catalytic states of the enzyme. Inhibition of TREX1 by lithium and sodium is consistent with alterations in the two-metalion phosphoryl-transfer mechanism proposed for the DnaQ family (Brautigam et al 1999a;Hamdan et al 2002b). According to this hypothesis, the metal ion located at site A would activate an hydroxyl nucleophile from a water molecule, via reducing the pKa of the water, and the ion located at site B would stabilize the transition state intermediate and the leaving group (Steitz and Steitz 1993;Hamdan et al 2002b).…”

Section: Discussionsupporting

confidence: 82%

“…Within this family, a high structural similarity of the active-site centers suggests a common catalytic mechanism. Through this mechanism, initially proposed for the Klenow exonuclease fragment of Escherichia coli, a water molecule is activated by the conserved histidine from the motif and by the magnesium (or manganese) at site A to form a hydroxide ion, which would carry out a nucleophilic attack on the phosphorus atom of the 39-terminal phosphodiester group, thereby contributing a 59-monophosphate oxygen to the mononucleotide product (Brautigam et al 1999a;Hamdan et al 2002b).…”

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confidence: 99%

“…The inhibition of TREX1 exerted by sodium has not been previously reported. Several structural enzymological studies performed on DNA exonucleases have contributed to a better understanding of the catalytic mechanism of these enzymes (Brautigam and Steitz 1998;Brautigam et al 1999a). However, no information is available on the inhibitory properties of monovalent ions, such as lithium and sodium.…”

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Structural and biochemical studies of TREX1 inhibition by metals. Identification of a new active histidine conserved in DEDDh exonucleases

et al. 2008

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TREX1 is the major exonuclease in mammalian cells, exhibiting the highest level of activity with a 39!59 activity. This exonuclease is responsible in humans for Aicardi-Goutières syndrome and for an autosomal dominant retinal vasculopathy with cerebral leukodystrophy. In addition, this enzyme is associated with systemic lupus erythematosus. TREX1 belongs to the exonuclease DEDDh family, whose members display low levels of sequence identity, while possessing a common fold and active site organization. For these exonucleases, a catalytic mechanism has been proposed that involves two divalent metal ions bound to the DEDD motif. Here we studied the interaction of TREX1 with the monovalent cations lithium and sodium. We demonstrate that these metals inhibit the exonucleolytic activity of TREX1, as measured by the classical gel method, as well as by a new technique developed for monitoring the real-time exonuclease reaction. The X-ray structures of the enzyme in complex with these two cations and with a nucleotide, a product of the exonuclease reaction, were determined at 2.1 Å and 2.3 Å , respectively. A comparison with the structures of the active complexes (in the presence of magnesium or manganese) explains that the inhibition mechanism is caused by the noncatalytic metals competing with distinct affinities for the two metal-binding sites and inducing subtle rearrangements in active centers. Our analysis also reveals that a histidine residue (His124), highly conserved in the DEDDh family, is involved in the activity of TREX1, as confirmed by mutational studies. Our results shed further light on the mechanism of activity of the DEDEh family of exonucleases.Keywords: exonucleases; TREX1; DEDDh family; structure; enzymatic activityThe 39!59 exonucleases play a crucial role in many biological processes by removing nucleotides at the 39 termini from DNA ends, which must then be remodeled in order to prevent the formation of aberrant structures. The 39!59 exonucleases were initially found as proofreading enzymes associated with DNA polymerases. However, in recent years several eukaryotic 39!59 exonucleases, such as p53, the Werner syndrome protein (WRN), and MRE11, 3 These two authors contributed equally to this work.

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Section: Discussionsupporting

confidence: 82%

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Structural and biochemical studies of TREX1 inhibition by metals. Identification of a new active histidine conserved in DEDDh exonucleases

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“…Second, our data do not exclude the possibility that the pro-S oxygen of the scissile bond coordinates a divalent metal ion, because we could not recover PARN activity using the A 3 s-S p isomeric substrate. Similarly, Brautigam and Steitz (17,32,33) could not detect the 3Ј-exonucleolytic activity of DNA pol I when they used an S p isomeric phosphorothioate oligonucleotide as substrate. Based on their structural analysis they proposed that the bulky sulfur atom on the sulfur-substituted substrate inactivated the enzyme by excluding cations in the 3Ј-exonucleolytic active site.…”

Section: Discussionmentioning

confidence: 99%

“…In the crystal structure (14,17,32,33) of the 3Ј-exonucleolytic active site of DNA pol I (Fig. 7), it was observed that the pro-S oxygen of the scissile bond coordinated two metal ions while an interaction between the pro-R oxygen and a divalent metal was not seen.…”

Section: Discussionmentioning

confidence: 99%

Coordination of Divalent Metal Ions in the Active Site of Poly(A)-specific Ribonuclease

Ren

Kirsebom

Virtanen

2004

Journal of Biological Chemistry

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Poly(A)-specific ribonuclease (PARN) is a highly poly(A)-specific 3-exoribonuclease that efficiently degrades mRNA poly(A) tails. PARN belongs to the DEDD family of nucleases, and four conserved residues are essential for PARN activity, i.e. Asp-28, Glu-30, Asp-292, and Asp-382. Here we have investigated how catalytically important divalent metal ions are coordinated in the active site of PARN. Each of the conserved amino acid residues was substituted with cysteines, and it was found that all four mutants were inactive in the pres Poly(A)-specific ribonuclease (PARN)1 is a highly poly(A)-specific 3Ј-exonuclease that efficiently degrades mRNA poly(A) tails (1-6). PARN is oligomeric, most likely consisting of three subunits (i.e. homotrimer) (6), and interacts with both the 3Ј-end-located poly(A) tail and the 5Ј-end cap structure during degradation (6 -9). The interaction with the 5Ј-end cap structure enhances the rate of degradation (6, 7, 9) and amplifies the processivity of PARN activity (9). The functional significance of PARN is still unknown, although its high specificity for poly(A) and interaction with the cap structure provide strong arguments for PARN being involved in mammalian mRNA poly(A) tail degradation and therefore could play important roles in mRNA decay and initiation of protein synthesis (6 -9).PARN belongs to the DEDD family of nucleases, and four conserved acidic amino acids characteristic of this family are present in PARN (5, 10 -12). These amino acids are Asp-28, Glu-30, Asp-292, and Asp-382 in human PARN. Site-directed mutagenesis has shown that these conserved residues are essential for catalytic activity and that they are required for the binding of divalent metal ions to PARN (13). Based on these observations it was proposed (13) that PARN utilizes the twometal ion mechanism for its catalysis, as suggested for the Escherichia coli 3Ј-exonuclease domain of DNA polymerase I (pol I) (14, 15), although the stoichiometry of divalent metal ion binding in the active site of PARN is not yet known. To fully understand the catalytic mechanism of PARN it is of fundamental importance to determine metal ion binding coordination and stoichiometry in its active site.Structural analyses by x-ray or NMR are powerful strategies to reveal the presence of divalent metal ions in the active sites of enzymes. However, despite the high resolution provided by these techniques, these methods frequently fail to unambiguously locate catalytically important divalent metal ions. Furthermore, even if the divalent metal ions are correctly located in the active site by structural analysis, it is still necessary to functionally confirm the interaction between the divalent metal ions and their ligands (16). Thus, other techniques are required to locate functionally important metal ions. This is of particular importance when structural data, as in the case of PARN, is not available. Sulfur substitutions of oxygen believed to be in direct contact with Mg 2ϩ ions have successfully been used to locate ligands in direct con...

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Crystallization of Protein– DNA Complexes

Pakotiprapha

Jeruzalmi

2009

Encyclopedia of Life Sciences

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Determination of atomic resolution structures of protein– deoxyribonucleic acid (DNA) complexes using X‐ray crystallography requires preparation of high‐quality crystalline specimens. The preparation of such samples remains an empirical endeavour, more art than science. Nonetheless, decades of experience have yielded a large body of practical strategies and techniques towards this end. Such efforts with protein–DNA complexes enjoy special advantages, but also involve control of a larger set of variables. High purity, chemical and conformational, preparations of both macromolecules are important. Crystallogenesis of protein–DNA complexes is favoured by careful choice of the sequence and the terminal bases and/or base pairs of the DNA target. Modern crystallization screens, which encapsulate the accumulated wisdom of prior investigations, serve as an excellent starting point for experimentation. Key Concepts An atomic level analysis of a macromolecule by X‐ray crystallography requires preparation of high‐quality crystals of the entity of interest. Preparation of crystalline specimens requires highly concentrated solutions of the molecules of interest. Success is favoured when high chemical and conformational purity has been achieved. Extensive experience in preparation of protein crystals can be applied to growth of protein–DNA crystals. Additionally, the length, composition and identity of the termini of the DNA molecule play significant roles in crystallization. Initial crystallization trials are set up using a concentrated stock of protein–DNA complex of interest with pre‐made formulations that sparsely sample crystallization space. Precipitation and crystal formation under these conditions are used to direct efforts for further crystallization experiments. The experiments are repeated until crystals suitable for X‐ray diffraction are obtained.

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Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' exonucleolytic active site of the Klenow fragment

Cited by 37 publications

References 0 publications

Structural and biochemical studies of TREX1 inhibition by metals. Identification of a new active histidine conserved in DEDDh exonucleases

Structural and biochemical studies of TREX1 inhibition by metals. Identification of a new active histidine conserved in DEDDh exonucleases

Coordination of Divalent Metal Ions in the Active Site of Poly(A)-specific Ribonuclease

Crystallization of Protein– DNA Complexes

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