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At least 325 records · Page 18Linked to original sources
PubMed1995

Base-pair opening and spermine binding--B-DNA features displayed in the crystal structure of a gal operon fragment: implications for protein-DNA recognition.

A sequence that is represented frequently in functionally important sites involving protein-DNA interactions is GTG/CAC, suggesting that the trimer may play a role in regulatory processes. The 2.5 A resolution structure of d(CGGTGG)/d(CCACCG), a part of the interior operator (OI, nucleotides +44 to +49) of the gal operon, co-crystallized with spermine, is described herein. The crystal packing arrangement in this structure is unprecedented in a crystal of B-DNA, revealing a close packing of columns of stacked DNA resembling a 5-stranded twisted wire cable. The final structure contains one hexamer duplex, 17 water molecules and 1.5 spermine molecules per crystallographic asymmetric unit. The hexamer exhibits base-pair opening and shearing at T.A resulting in a novel non-Watson-Crick hydrogen-bonding scheme between adenine and thymine in the GTG region. The ability of this sequence to adopt unusual conformations in its GTG region may be a critical factor conferring sequence selectivity on the binding of Gal repressor. In addition, this is the first conclusive example of a crystal structure of spermine with native B-DNA, providing insight into the mechanics of polyamine-DNA binding, as well as possible explanations for the biological action of spermine.

Base Composition↗
PubMed2002

TraY DNA recognition of its two F factor binding sites.

F factor TraY, a ribbon-helix-helix DNA-binding protein, performs two roles in bacterial conjugation. TraY binds the F origin of transfer (oriT) to promote nicking of plasmid DNA prior to conjugative transfer. TraY also binds the P(Y) promoter to up-regulate tra gene expression. The two plasmid regions bound by TraY share limited sequence identity, yet TraY binds them with similar affinities. TraY recognition of the two sites was first probed using in vitro footprinting methods. Hydroxyl radical footprinting at both oriT and P(Y) sites indicated that bound TraY protected the DNA backbone bordering three adjacent DNA subsites. Analytical ultracentrifugation results for TraY:oligonucleotide complexes were consistent with two of these subsites being bound cooperatively, and the third being occupied at higher TraY concentrations. Methylation protection and interference footprinting identified several guanine bases contacted by or proximal to bound TraY, most located within these subsites. TraY affinity for variant oriT sequences with base substitutions at or near these guanine bases suggested that two of the three subsites correspond to high-affinity, cooperatively bound imperfect inverted GA(G/T)A repeats. Altering the spacing or orientation of these sites reduced binding. TraY mutant R73A failed to protect two symmetry-related oriT guanine bases in these repeats from methylation, identifying possible direct TraY-DNA contacts. The third subsite appears to be oriented as an imperfect direct repeat with its adjacent subsite, although base substitutions at this subsite did not reduce binding. Although unusual for ribbon-helix-helix proteins, this binding site arrangement occurs at both F TraY sites, consistent with it being functionally relevant.

Base Sequence↗
PubMed2001

Toward rules for 1:1 polyamide:DNA recognition.

Polyamides composed of four amino acids, imidazole (Im), pyrrole (Py), hydroxypyrrole (Hp), and beta-alanine (beta), are synthetic ligands that form highly stable complexes in the minor groove of DNA. Although specific pairing rules within the 2:1 motif can be used to distinguish the four Watson. Crick base pairs, a comparable recognition code for 1:1 polyamide:DNA complexes had not been described. To set a quantitative baseline for the field, the sequence specificities of Im, Py, Hp, and beta for the four Watson. Crick base pairs were determined for two polyamides, Im-beta-ImPy-beta-Im-beta-ImPy-beta-Dp (1, for Im, Py, and beta) and Im-beta-ImHp-beta-Im-beta-ImPy-beta-Dp (2, for Hp), in a 1:1 complex within the DNA sequence context 5'-AAAGAGAAGAG-3'. Im residues do not distinguish G,C from A,T but bind all four base pairs with high affinity. Py and beta residues exhibit > or = 10-fold preference for A,T over G,C base pairs. The Hp residue displays a unique preference for a single A.T base pair with an energetic penalty.

Base Sequence↗
PubMed1999

Crystal structure of an IRF-DNA complex reveals novel DNA recognition and cooperative binding to a tandem repeat of core sequences.

There has been growing interest in the role of the IRF (interferon regulatory factor) family of transcription factors in the regulation of immune responses, cytokine signaling, and oncogenesis. These members are characterized by their well-conserved DNA binding domains at the N-terminal regions. Here we report the 2.2 A resolution crystal structure of the DNA binding domain of one such family member, IRF-2, bound to DNA. The structure reveals its recognition sequence, AANNGAAA (here, recognized bases are underlined and in bold, and N indicates any base), and its cooperative binding to a tandem repeat of the GAAA core sequence induced by DNA structure distortions. These facts explain well the diverse binding properties of the IRF family members, which bind to both single and tandemly repeated sequences. Furthermore, we also identified the 'helix-hairpin-strand motif' at the C terminus of the recognition helix as a metal binding site that is commonly found in certain classes of DNA-interactive proteins. Our results provide new insights into the structure and function of this family of transcription factors.

Amino Acid Sequence↗
PubMed1995

The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site.

The hypoxia-inducible factor-1 (HIF-1) was first described as a DNA binding activity that specifically recognizes an 8 bp motif known to be essential for hypoxia-inducible erythropoietin gene transcription. Subsequently HIF-1 activity has also been found in cell lines which do not express erythropoietin, suggesting that HIF-1 is part of a widespread oxygen sensing mechanism. In electrophoretic mobility shift assays HIF-1 DNA binding activity is only detectable in nuclear extracts of cells cultivated in a low oxygen atmosphere. In addition to HIF-1, a constitutive DNA binding activity also specifically binds the HIF1 probe. Here we report that CRE and AP1 oligonucleotides efficiently competed for binding of the HIF1 probe to this constitutive factor, whereas HIF-1 activity itself remained unaffected. Monoclonal antibodies raised against the CRE binding factors ATF-1 and CREB-1 supershifted the constitutive factors ATF-1 and CREB-1 supershifted the constitutive factor, while Jun and Fos family members, which constitute the AP-1 factor, were immunologically undetectable. Recombinant ATF-1 and CREB-1 proteins bound HIF1 probes either as homodimers or as heterodimers, indicating a new binding specificity for ATF-1/CREB-1. Finally, reporter gene assays in HeLa cells treated with either a cAMP analogue or a phorbol ester suggest that the PKA, but not the PKC signalling pathway is involved in oxygen sensing.

Activating Transcription Factor 1↗
PubMed1996

Mismatch DNA recognition protein from an extremely thermophilic bacterium, Thermus thermophilus HB8.

The mutS gene, implicated in DNA mismatch repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 819-amino acid protein with a molecular mass of 91.4 kDa. Its predicted amino acid sequence showed 56 and 39% homology with Escherichia coli MutS and human hMsh2 proteins, respectively. The T.thermophilus mutS gene complemented the hypermutability of the E.coli mutS mutant, suggesting that T.thermophilus MutS protein was active in E.coli and could interact with E.coli MutL and/or MutH proteins. The T.thermophilus mutS gene product was overproduced in E.coli and then purified to homogeneity. Its molecular mass was estimated to be 91 kDa by SDS-PAGE but approx. 330 kDa by size-exclusion chromatography, suggesting that T.thermophilus MutS protein was a tetramer in its native state. Circular dichroic measurements indicated that this protein had an alpha-helical content of approx. 50%, and that it was stable between pH 1.5 and 12 at 25 degree C and was stable up to 80 degree C at neutral pH. Thermus thermophilus MutS protein hydrolyzed ATP to ADP and Pi, and its activity was maximal at 80 degrees C. The kinetic parameters of the ATPase activity at 65 degrees C were Km = 130 microM and Kcat = 0.11 s(-1). Thermus thermophilus MutS protein bound specifically with G-T mismatched DNA even at 60 degrees C.

Adenosine Triphosphatases↗
PubMed2000

DNA recognition, strand selectivity, and cleavage mode during integrase family site-specific recombination.

We have probed the association of Flp recombinase with its DNA target using protein footprinting assays. The results are consistent with the domain organization of the Flp protein and with the general features of the protein-DNA interactions revealed by the crystal structures of the recombination intermediates formed by Cre, the Flp-related recombinase. The similarity in the organization of the Flp and Cre target sites and in their recognition by the respective recombinases implies that the overall DNA-protein geometry during strand cleavage in the two systems must also be similar. Within the functional recombinase dimer, it is the interaction between two recombinase monomers bound on either side of the strand exchange region (or spacer) that provides the allosteric activation of a single active site. Whereas Cre utilizes the cleavage nucleophile (the active site tyrosine) in cis, Flp utilizes it in trans (one monomer donating the tyrosine to its partner). By using synthetic Cre and Flp DNA substrates that are geometrically restricted in similar ways, we have mapped the positioning of the active and inactive tyrosine residues during cis and trans cleavage events. We find that, for a fixed substrate geometry, Flp and Cre cleave the labile phosphodiester bond at the same spacer end, not at opposite ends. Our results provide a model that accommodates local heterogeneities in peptide orientations in the two systems while preserving the global functional architecture of the reaction complex.

Amino Acid Sequence↗
PubMed2002

Pressure induces folding intermediates that are crucial for protein-DNA recognition and virus assembly.

Protein-nucleic acid interactions are crucial for a variety of fundamental biological processes such as replication, transcription, restriction, translation and virus assembly. The molecular basis of protein-DNA and protein-RNA recognition is deeply related to the thermodynamics of the systems. We review here how protein-nucleic acid interactions can be approached in the same way as protein-protein interactions involved in protein folding and protein assembly, using hydrostatic pressure as the primary tool and employing several spectroscopic techniques, especially fluorescence, circular dichroism and high-resolution nuclear magnetic resonance. High pressure has the unique property of stabilizing partially folded states or molten-globule states of a protein. The competition between correct folding and misfolding, which in many proteins leads to formation of insoluble aggregates is an important problem in the biotechnology industry and in human diseases such as amyloidosis, Alzheimer's, prion and tumor diseases. The pressure studies reveal that a gradient of partially folded (molten globule) conformations is present between the unfolded and fully folded structure of several bacteria, plant and mammalian viruses. Using pressure, we have detected the presence of a ribonucleoprotein intermediate, where the coat protein is partially unfolded but bound to RNA. These intermediates are potential targets for antiviral compounds. Pressure studies on viruses have direct biotechnological applications. The ability of pressure to inactivate viruses has been evaluated with a view toward the applications of vaccine development and virus sterilization. Recent studies demonstrate that pressure causes virus inactivation while preserving the immunogenic properties. There is substantial evidence that a high-pressure cycle traps a virus in the 'fusion intermediate state', not infectious but highly immunogenic.

Amyloid↗
PubMed2004

Evolution of divergent DNA recognition specificities in VDE homing endonucleases from two yeast species.

Homing endonuclease genes (HEGs) are mobile DNA elements that are thought to confer no benefit to their host. They encode site-specific DNA endonucleases that perpetuate the element within a species population by homing and disseminate it between species by horizontal transfer. Several yeast species contain the VMA1 HEG that encodes the intein-associated VMA1-derived endonuclease (VDE). The evolutionary state of VDEs from 12 species was assessed by assaying their endonuclease activities. Only two enzymes are active, PI-ZbaI from Zygosaccharomyces bailii and PI-ScaI from Saccharomyces cariocanus. PI-ZbaI cleaves the Z.bailii recognition sequence significantly faster than the Saccharomyces cerevisiae site, which differs at six nucleotide positions. A mutational analysis indicates that PI-ZbaI cleaves the S.cerevisiae substrate poorly due to the absence of a contact that is analogous to one made in PI-SceI between Gln-55 and nucleotides +9/+10. PI-ZbaI cleaves the Z.bailii substrate primarily due to a single base-pair substitution (A/T+5 --> T/A+5). Structural modeling of the PI-ZbaI/DNA complex suggests that Arg-331, which is absent in PI-SceI, contacts T/A+5, and the reduced activity observed in a PI-ZbaI R331A mutant provides evidence for this interaction. These data illustrate that homing endonucleases evolve altered specificity as they adapt to recognize alternative target sites.

Amino Acid Sequence↗
PubMed1998

Role of protein-induced bending in the specificity of DNA recognition: crystal structure of EcoRV endonuclease complexed with d(AAAGAT) + d(ATCTT).

The crystal structure of EcoRV endonuclease has been determined at 2. 1 A resolution complexed to two five-base-pair DNA duplexes each containing the cognate recognition half-site. The highly localized 50 degrees bend into the major groove seen at the center TA-step of the continuous GATATC site is preserved in this discontinuous DNA complex lacking the scissile phosphates. Thus, this crystal structure provides evidence that covalent constraints associated with a continuous target site are not essential to enzyme-induced DNA bending, even when these constraints are removed directly at the locus of the bend. The scissile phosphates are also absent in the crystal structure of EcoRV bound to the non-specific site TCGCGA, which shows a straight B-like conformation. We conclude that DNA bending by EcoRV is governed only by the sequence and is not influenced by the continuity of the phosphodiester backbone. Together with other data showing that cleavable non-cognate sites are bent, these results indicate that EcoRV bends non-cognate sites differing by one or two base-pairs from GATATC, but does not bend non-specific sites that are less similar. Structural and thermodynamic considerations suggest that the sequence-dependent energy cost of DNA bending is likely to play an important role in determining the specificity of EcoRV. This differential cost is manifested at the binding step for bent non-cognate sequences and at the catalytic step for unbent non-specific sequences.

Base Sequence↗
PubMed1999

A novel paired domain DNA recognition motif can mediate Pax2 repression of gene transcription.

The paired domain (PD) is an evolutionarily conserved DNA-binding domain encoded by the Pax gene family of developmental regulators. The Pax proteins are transcription factors and are involved in a variety of processes such as brain development, patterning of the central nervous system (CNS), and B-cell development. In this report we demonstrate that the zebrafish Pax2 PD can interact with a novel type of DNA sequences in vitro, the triple-A motif, consisting of a heptameric nucleotide sequence G/CAAACA/TC with an invariant core of three adjacent adenosines. This recognition sequence was found to be conserved in known natural Pax5 repressor elements involved in controlling the expression of the p53 and J-chain genes. By identifying similar high affinity binding sites in potential target genes of the Pax2 protein, including the pax2 gene itself, we obtained further evidence that the triple-A sites are biologically significant. The putative natural target sites also provide a basis for defining an extended consensus recognition sequence. In addition, we observed in transformation assays a direct correlation between Pax2 repressor activity and the presence of triple-A sites. The results suggest that a transcriptional regulatory function of Pax proteins can be modulated by PD binding to different categories of target sequences.

5' Untranslated Regions↗
PubMed2003

Site-directed mutagenesis reveals the thermodynamic requirements for single-stranded DNA recognition by the telomere-binding protein Cdc13.

The essential Saccharomyces cerevisiae protein Cdc13 binds the conserved single-stranded overhang at the end of telomeres and mediates access of protein complexes involved in both end-capping and telomerase activity. The single-stranded DNA-binding domain (ssDBD) of Cdc13 exhibits both high affinity (K(d) of 3 pM) and sequence specificity for the GT-rich sequences present at yeast telomeres. We have used the ssDBD of Cdc13 to understand the sequence-specific recognition of extended single-stranded DNA (ssDNA). The recent structure of the Cdc13 DNA-binding domain revealed that ssDNA is recognized by a large protein surface containing an oligonucleotide/oligosaccharide-binding fold (OB-fold) augmented by an extended 30-amino acid loop. Contacts to ssDNA occur via a contiguous surface of aromatic, hydrophobic, and basic residues. A complete alanine scan of the binding interface has been used to determine the contribution of each contacting side chain to binding affinity. Substitution of any aromatic or hydrophobic residue at the interface was deleterious to binding (20 to >700-fold decrease in binding affinity), while tolerance for replacement of basic residues was observed. The important aromatic and hydrophobic contacts are spread throughout the extended interface, indicating that the entire surface is both structurally and thermodynamically required for binding. While all of these contacts are important, several of the individual alanine substitutions that abolish binding cluster to one region of the protein surface. This region is vital for recognition of four bases at the 5' end of the DNA and constitutes a "hotspot" of binding affinity.

Binding Sites↗
PubMed1996

Participation of water in Hin recombinase--DNA recognition.

The participation of water molecules in the interaction between the Hin recombinase and its operator DNA has been detected by analysis of the dissociation constant in the presence of varying concentrations of neutral solutes and cosolvents. The dissociation constant as measured by gel mobility shift assays increased as the concentration of dimethyl sulfoxide, glycerol, sucrose, or polyethylene glycol was increased. Osmotic pressure is the only property that correlates with the change in the dissociation constant for all compounds. This data indicates that binding of a small population of water molecules accompanies formation of the Hin-DNA complex, and points to a novel role for solvent molecules in assisting site specific interaction between DNA-binding proteins and their cognate DNA sequence.

Bacterial Proteins↗