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D Suck

Publications and source records attributed to D Suck.

At least 19 recordsLinked to original sources
PubMed1998

Recognition of single-stranded DNA by nuclease P1: high resolution crystal structures of complexes with substrate analogs.

The reaction mechanism of nuclease P1 from Penicillium citrinum has been investigated using single-stranded dithiophosphorylated di-, tetra-, and hexanucleotides as substrate analogs. The complexes crystallize in tetragonal and orthorhombic space groups and have been solved by molecular replacement. The high resolution structures give a clear picture of base recognition by P1 nuclease at its two nucleotide-binding sites, especially the 1.8 A structure of a P1-tetranucleotide complex which can be considered a P1-product complex. The observed binding modes are in agreement with a catalytic mechanism where the two closely spaced zinc ions activate the attacking water while the third, more exposed zinc ion stabilizes the leaving 03' oxyanion. Stacking as well as hydrogen bonding interactions with the base 5' to the cleaved phosphodiester bond are important elements of substrate binding and recognition. Modelling of a productive P1-substrate complex based on the solved structures suggests steric hindrance as the likely reason for the resistance of Rp-phosphorothioates and phosphorodithioates. Differences with the highly homologous nuclease S1 from Aspergillus oryzae are discussed.

Catalysis↗
PubMed1997

Prokaryotic 5'-3' exonucleases share a common core structure with gamma-delta resolvase.

The three dimensional crystal structure of T5 5'-3' exonuclease was compared with that of two other members of the 5'-3' exonuclease family: T4 ribonuclease H and the N-terminal domain of Thermus aquaticus DNA polymerase I. Though these structures were largely similar, some regions of these enzymes show evidence of significant molecular flexibility. Previous sequence analysis had suggested the existence of a helix-hairpin-helix motif in T5 exonuclease, but a distinct, though related structure is actually found to occur. The entire T5 exonuclease structure was then compared with all the structures in the complete Protein Data Bank and an unexpected similarity with gamma-delta (gamma delta) resolvase was observed. 5'-3' exonucleases and gamma delta resolvase are enzymes involved in carrying out quite different manipulations on nucleic acids. They appear to be unrelated at the primary sequence level, yet the fold of the entire catalytic domain of gamma delta resolvase is contained within that of the 5'-3'exonuclease. Different large-scale helical structures are used by both families to form DNA binding sites.

Amino Acid Sequence↗
PubMed1997

Slight sequence variations of a common fold explain the substrate specificities of tRNA-guanine transglycosylases from the three kingdoms.

tRNA-guanine transglycosylases (TGTs) are the enzymes catalyzing the base exchange required for the synthesis of the modified bases derived from 7-deazaguanine in prokaryotic, archaebacterial, and eukaryotic tRNAs. Unlike the eukaryotic and archaebacterial enzymes, the prokaryotic TGTs have been clearly identified and highly characterized both biochemically and structurally. The recent occurrence in sequence databases of archaebacterial and eukaryotic proteins homologous to the prokaryotic TGTs reveals that all TGTs unexpectedly adopt a common fold. Observed sequence variations at the active site correlate well with their specificities for the various 7-deazaguanine derivatives and the total conservation of the catalytic residues strongly favors a common catalytic mechanism for all TGTs.

Amino Acid Sequence↗
PubMed1997

The crystal structure of an intact human Max-DNA complex: new insights into mechanisms of transcriptional control.

BACKGROUND: Max belongs to the basic helix-loop-helix leucine zipper (bHLHZ) family of transcription factors. Max is able to form homodimers and heterodimers with other members of this family, which include Mad, Mxi1 and Myc; Myc is an oncoprotein implicated in cell proliferation, differentiation and apoptosis. The homodimers and heterodimers compete for a common DNA target site (the E box) and rearrangement amongst these dimer forms provides a complex system of transcriptional regulation. Max is also regulated by phosphorylation at a site preceding the basic region. We report here the first crystal structure of an intact bHLHZ protein bound to its target site. RESULTS: The X-ray crystal structure of the intact human Max protein homodimer in complex with a 13-mer DNA duplex was determined to 2.8 A resolution and refined to an R factor of 0.213. The C-terminal domains in both chains of the Max dimer are disordered. In contrast to the DNA observed in complex with other bHLH and bHLHZ proteins, the DNA in the Max complex is bent by about 25 degrees, directed towards the protein. Intimate contacts with interdigitating sidechains give rise to the formation of tetramers in the crystal. CONCLUSIONS: The structure confirms the importance of the HLH and leucine zipper motifs in dimerization as well as the mode of E box recognition which was previously analyzed by X-ray crystallography of shortened constructs. The disorder observed in the C-terminal domain suggests that contacts with additional protein components of the transcription machinery are necessary for ordering the secondary structure. The tetramers seen in the crystal are consistent with the tendency of Max and other bHLHZ and HLH proteins to form higher order oligomers in solution and may play a role in DNA looping. The location of the two phosphorylation sites at Ser1 and Ser10 (the latter is the N-cap of the basic helix) suggests how phosphorylation could disrupt DNA binding.

Amino Acid Sequence↗
PubMed1997

The bifunctional protein DCoH modulates interactions of the homeodomain transcription factor HNF1 with nucleic acids.

The hepatocyte nuclear factor-1 (HNF1) is a homeodomain transcription factor that binds DNA as a dimer. HNF1 dimers associate with two molecules of DCoH, a bifunctional protein that also has an enzymatic function in the tetrahydrobiopterin regeneration, to form stable heterotetramers also capable of DNA binding. Employing purified, recombinant HNF1, HNF1/DCoH heterotetramers and DCoH homotetramers we investigated whether DCoH affects interactions of HNF1 with nucleic acids. Although we detected no direct binding of DCoH to DNA or RNA, DCoH stabilized HNF1/DNA complexes and promoted interactions with sub-optimal DNA target sequences such as the human alpha1-antitrypsin TATA box region. Importantly, we also observed interactions of HNF1 with RNA, but these interactions were completely abolished when HNF1 was complexed with DCoH. Interestingly, DCoH retains its enzymatic activity while complexed with HNF1. Our results document intermolecular regulation of HNF1 binding to nucleic acids by DCoH.

Animals↗
PubMed1996

Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase reveal aspartate 102 as the active site nucleophile.

Procaryotic tRNA-guanine transglycosylase (TGT) catalyzes the posttranscriptional base exchange of the queuine precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine at the wobble position of tRNAs specific for Asn, Asp, His, and Tyr. The X-ray structures of Zymomonas mobilis TGT and of its complex with preQ1 [Romier, C., Reuter, K., Suck, D., & Ficner, R. (1996) EMBO J. 15, 2850-2857] have revealed a specific preQ1 binding pocket and allowed a proposal for tRNA binding and recognition. We have used band-shift experiments in denaturing conditions to study the enzymatic reaction performed by TGT. The presence of shifted protein bands after incubation with tRNA followed by protein denaturation indicates a reaction mechanism involving a covalent intermediate. Inspection of the X-ray structures and comparison of the different procaryotic TGT sequences highlighted the conserved aspartate 102 as the most likely nucleophile. Mutation of this residue into alanine by site-directed mutagenesis leads to an inactive mutant unable to form a covalent intermediate with tRNA, proving that aspartate 102 is the active site nucleophile in TGT. To investigate the recognition of the wobble guanine in the preQ1 binding pocket, we mutated aspartate 156, the major recognition element for preQ1, into alanine and tyrosine. Both mutants are inactive in producing the final product, but the mutant D156A is able to form the covalent intermediate with tRNA in the first step of the reaction mechanism in comparable amounts to wild-type protein. Therefore, the binding of the wobble guanine in the preQ1 binding pocket is required for the cleavage of the glycosidic bond. The three mutants were crystallized and their X-ray structures determined. The mutants display only subtle changes to the wild-type protein, confirming that the observed biochemical results are due to the chemical substitutions rather than structural rearrangements.

Aspartic Acid↗
PubMed1996

Location of the active site and proposed catalytic mechanism of pterin-4a-carbinolamine dehydratase.

Based on the recently solved three-dimensional structure of pterin-4a-carbinolamine dehydratase from rat/human liver the involvement of the proposed active-site residues Glu57, Asp60, His61, His62, Tyr69, His79, Arg87 and Asp88 was examined by site-directed mutagenesis. Most of the mutants showed reduced activity, and only the Glu57-->Ala mutant and the His61-->Ala, His62-->Ala double mutant were fully devoid of activity. The dissociation constants of quinonoid 6,6-dimethyl-7,8-dihydropterin were significantly increased for binding to the Glu57-->Ala, His61-->Ala, His62-->Ala single mutants and the His61-->Ala, His62-->Ala double mutant, confirming that His61 and His62 are essential for substrate binding and catalysis. The mechanism of dehydration is proposed to involve base catalysis at the N(5)-H group of the substrate by His61.

Amino Acid Sequence↗
PubMed1996

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

THE 5'-exonucleases are enzymes that are essential for DNA replication and repair. As well as their exonucleolytic action, removing nucleotides from the 5'-end of nucleic acid molecules such as Okazaki fragments, many 5'-3'-exonucleases have been shown to possess endonucleolytic activities. T5 5'-3'-exonuclease shares many similarities with the amino terminal of eubacterial DNA polymerases, although, unlike eubacteria, phages such as T5, T4 and T7 express polymerase and 5'-exonuclease proteins from separate genes. Here we report the 2.5-A crystal structure of the phage T5 5'-exonuclease, which reveals a helical arch for binding DNA. We propose a model consistent with a threading mechanism in which single-stranded DNA could slide through the arch, which is formed by two helices, one containing positively charged, and the other hydrophobic, residues. The active site is at the base of the arch, and contains two metal-binding sites.

Binding Sites↗
PubMed1996

Structure and function of PCD/DCoH, an enzyme with regulatory properties.

The bifunctional protein PCD/DCoH is both an enzyme involved in the phenylalanine hydroxylation system and a transcription coactivator forming a 2:2 heterotetrameric complex with the nuclear transcription factor HNF1. The discovery of a bacterial homologue and the expression pattern during Xenopus embryogenesis suggest a regulatory function not only restricted to HNF1. The crystal structures of the tetrameric rat and the dimeric bacterial PCD/DCoH have led to the proposal of substrate and HNF1 binding sites. The saddle-shaped beta-sheet surfaces of the DCoH dimers likely represent binding sites for as yet unknown macromolecular interaction partners. Possible mechanisms for DCoH-induced transcriptional regulation are discussed in the light of the three-dimensional structures.

Amino Acid Sequence↗
PubMed1996

Crystal structure of tRNA-guanine transglycosylase: RNA modification by base exchange.

tRNA-guanine transglycosylases (TGT) are enzymes involved in the modification of the anticodon of tRNAs specific for Asn, Asp, His and Tyr, leading to the replacement of guanine-34 at the wobble position by the hypermodified base queuine. In prokaryotes TGT catalyzes the exchange of guanine-34 with the queuine (.)precursor 7-aminomethyl-7-deazaguanine (preQ1). The crystal structure of TGT from Zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 19% at 1.85 angstrom resolution. The structure consists of an irregular (beta/alpha)8-barrel with a tightly attached C-terminal zinc-containing subdomain. The packing of the subdomain against the barrel is mediated by an alpha-helix, located close to the C-terminus, which displaces the eighth helix of the barrel. The structure of TGT in complex with preQ1 suggests a binding mode for tRNA where the phosphate backbone interacts with the zinc subdomain and the U33G34U35 sequence is recognized by the barrel. This model for tRNA binding is consistent with a base exchange mechanism involving a covalent tRNA-enzyme intermediate. This structure is the first example of a (beta/alpha)-barrel protein interacting specifically with a nucleic acid.

Amino Acid Sequence↗
PubMed1996

Purification, crystallization, and preliminary x-ray diffraction studies of tRNA-guanine transglycosylase from Zymomonas mobilis.

The tRNA modifying enzyme tRNA-gnanine transglycosylase (Tgt) catalyzes the exchange of guanine in the first position of the anticodon with the quenine precursor 7-aminomethyl-7-deazagnanine. Tgt from Zymomonas mobilis has been purified by crystallization and further recrystallized to obtain single crystals suitable for X-ray diffraction studies. Crystals were grown by vapor diffusion/gel crystallization methods using PEG 8,000 as precipitant. Macroseeding techniques were employed to produce large single crystals. The crystals of Tgt belong to the monoclinic space group C2 with cell constants a = 92.1 A, b = 65.1 A, c = 71.9 A, and beta = 97.5 degrees and contain one molecule per asymmetric unit. A complete diffraction data set from one native crystal has been obtained at 1.85 A resolution.

Chromatography, Ion Exchange↗
PubMed1995

Three-dimensional structure of the bifunctional protein PCD/DCoH, a cytoplasmic enzyme interacting with transcription factor HNF1.

The bifunctional protein pterin-4a-carbinolamine dehydratase (PCD)/dimerization cofactor of HNF1 (DCoH) is a cytoplasmic enzyme involved in the tetrahydrobiopterin regeneration and is found in complex with the transcription factor HNF1 in liver cell nuclei. An atypical hyperphenylalaninemia and the depigmentation disorder vitiligo are related to a deficiency of PCD/DCoH activity. The crystal structure of PCD/DCoH was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 20.5% at 2.7 A resolution. The single domain monomer comprises three alpha-helices packed against one side of a four-stranded, antiparallel beta-sheet. The functional enzyme is a homo-tetramer of 222 symmetry where each of the monomers contributes one helix to a central four helix bundle. In the tetramer two monomers form an eight-stranded, antiparallel beta-sheet with six helices packing against it from one side. The concave, hydrophobic surface of the eight-stranded beta-sheet with its two protruding loops at either end is reminiscent of the saddle-like shape seen in the TATA-box binding protein. PCD/DCoH binds as a dimer to the helical dimerization domain of dimeric HNF1 forming a hetero-tetramer possibly through a mixed four helix bundle.

Amino Acid Sequence↗
PubMed1995

Sequence-dependent bending propensity of DNA as revealed by DNase I: parameters for trinucleotides.

Structural parameters characterizing the bending propensity of trinucleotides were deduced from DNase I digestion data using simple probabilistic models. In contrast to dinucleotide-based models of DNA bending and/or bendability, the trinucleotide parameters are in good agreement with X-ray crystallographic data on bent DNA. This improvement may be due to the fact that the trinucleotide model incorporates more sequence context information than do dinucleotide-based descriptions.

Base Sequence↗
PubMed1995

Crystallization and preliminary crystallographic studies of recombinant dimerization cofactor of transcription factor HNF1/pterin-4 alpha-carbinolamine dehydratase from liver.

The bi-functional protein dimerization cofactor of HNF1 (DCoH)/pterin-4 alpha-carbinolamine dehydratase (PCD) is found in liver cell nuclei bound to the transcription factor hepatocyte nuclear factor 1 (HNF1) as well as in the cytoplasm acting as an enzyme involved in the phenylalanine hydroxylation system. Deficiency of DCoH/PCD activity in liver causes an atypical hyperphenylalaninemia and deficiency in human epidermis is related to the depigmentation disorder vitiligo. DCoH/PCD from rat liver, which is identical to the human protein, was expressed in E. coli, purified to homogeneity and crystallized. The crystals belong to the trigonal space group P3(1)21 (or P3(2)21) with unit cell dimensions of a = b = 106.2 A, c = 197.1 A. Native crystals diffract to a resolution of 2.5 A.

Animals↗
PubMed1995

Trinucleotide models for DNA bending propensity: comparison of models based on DNaseI digestion and nucleosome packaging data.

DNaseI digestion studies (Brukner et al, EMBO J 14, 1812-1818 1995) and nucleosomebinding data (Satchwell et al, J. Mol. Biol. 191, 639-659 1986, Goodsell and Dickerson, Nucleic trinucleotides. A detailed comparison of the two models suggests that while both of them represent improvements with respect to dinucleotide based descriptions, the individual trinucleotide parameters are not highly correlated (linear correlation coefficient is 0.53), and a number of motifs such as TA-elements and CCA/TGG motifs are more realistically described in the DNaseI-based model. This may be due to the fact that the DNaseI-based model does not rely on a static geometry but rather captures a dynamic ability of ds DNA to bend towards the major grove. Future refinement of both models of both models on larger experimental data sets is expected to further improve the prediction of macroscopic DNA-curvature.

DNA↗
PubMed1995

Mutational analysis of DNase I-DNA interactions: design, expression and characterization of a DNase I loop insertion mutant with altered sequence selectivity.

A mutant of bovine pancreatic DNase I containing two additional residues in a loop next to C173 has been expressed in Escherichia coli, purified and characterized biochemically. Modelling studies suggest that the inserted arginine and glutamate side chains of the modified loop sequence C173-R-E-G-T-V176 could contact the bases 3' to the cleaved bond in the major groove of a bound DNA, and that up to 10 bp could interact with the enzyme and potentially influence its cutting rate. The loop insertion mutant has an 800-fold lower specific activity than wild-type and shows overall cleavage characteristics similar to bovine pancreatic DNase I. Compared with the wild-type enzyme, the mutant shows a strongly enhanced preference for cutting the inverted repeat: (formula: see text) or close variants thereof. Unexpectedly for a minor groove binding protein, the preferred cutting sites in opposite strands are staggered by 1 bp in the 5' direction, causing the cleavage of a TA and a TT step, respectively. This finding demonstrates that the sequence context is relatively more important for the cutting frequency than the nature of the dinucleotide step of the cleaved bond, and clearly shows that base recognition is involved in determining the sequence selectivity of the mutant. The importance of the sequence 5' to the cleaved bond for the cutting rate suggests that the additional major groove contacts may require a distortion of the DNA associated with a higher energy barrier, resulting in an increased selectivity for flexible DNA sequences and a lower overall activity of the mutant enzyme.

Animals↗
PubMed1994

DNA-protein interactions. Flip out and modify.

The crystal structure of a complex between a methyltransferase and DNA shows that, remarkably, the target cytosine base is swung out of the double helix and located next to the enzyme's S-adenosyl-L-homocysteine cofactor.

Base Sequence↗