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At least 577 records · Page 32Linked to original sources
PubMed1998

Molecular genetic analysis of transposase-end DNA sequence recognition: cooperativity of three adjacent base-pairs in specific interaction with a mutant Tn5 transposase.

Transposition of Tn5 and IS50 requires the specific binding of transposase (Tnp) to the end inverted repeats, the outside end (OE) and the inside end (IE). OE and IE have 12 identical base-pairs and seven non-identical base-pairs. Previously we described the isolation of a Tnp mutant, EK54, that shows an altered preference for OE versus IE compared to wild-type (wt) Tnp. EK54 enhances OE recognition and decreases IE recognition both in DNA binding and in overall transposition. Here we report that base-pairs 10, 11 and 12 of the OE are critical for the specific recognition by EK54 Tnp. These three adjacent base-pairs act cooperatively; all three must be present in order for EK54 Tnp to interact very favorably with the end DNA. The existence of only one or two of these three base-pairs decreases binding of EK54 Tnp. The combined use of EK54 Tnp and a new OE/IE mosaic end sequence containing the OE base-pairs 10, 11 and 12 gives rise to an extraordinarily high transposition frequency. Just as the Tnp is a multifunctional protein, the nucleotides in the 19 bp Tn5 ends also affect other functions besides Tnp binding. Furthermore, the fact that we were able to isolate end sequence variants that transpose at a higher frequency than the natural ends (OE and IE) with wt Tnp reveals yet another way in which the wt transposition frequency is depressed, i.e. by keeping the end sequences suboptimal.

Base Sequence↗
PubMed1995

Mechanisms of DNA damage recognition in mammalian nucleotide excision repair.

The ability of nucleotide excision repair (NER) to process multiple forms of DNA damage is highly dependent on the precision by which DNA modifications are located in the genome. Studies of mammalian NER have shown that this system eliminates a wide range of chemically and structurally distinct DNA lesions whereby some types of damage are repaired at higher rates than others. Although the biochemical basis for this broad but heterogeneous response to DNA damage is poorly understood, recent discoveries in closely related areas of DNA metabolism indicate that selectivity for specific sites is achieved through the assembly of nucleoprotein complexes, in which DNA is frequently bent and unwound. In many cases, selectivity may be further enhanced by the action of specialized DNA helicases. These principles in protein-DNA recognition suggest a hypothetical mechanism of damage recognition that accounts for the wide substrate range of mammalian NER and also accommodates its preference for specific DNA lesions.

Animals↗
PubMed2003

Imidazopyridine/Pyrrole and hydroxybenzimidazole/pyrrole pairs for DNA minor groove recognition.

The DNA binding properties of fused heterocycles imidazo[4,5-b]pyridine (Ip) and hydroxybenzimidazole (Hz) paired with pyrrole (Py) in eight-ring hairpin polyamides are reported. The recognition profile of Ip/Py and Hz/Py pairs were compared to the five-membered ring pairs Im/Py and Hp/Py on a DNA restriction fragment at four 6-base pair recognition sites which vary at a single position 5'-TGTNTA-3', where N = G, C, T, A. The Ip/Py pair distinguishes G.C from C.G, T.A, and A.T, and the Hz/Py pair distinguishes T.A from A.T, G.C, and C.G, affording a new set of heterocycle pairs to target the four Watson-Crick base pairs in the minor groove of DNA.

Base Sequence↗
PubMed2003

Critical DNA damage recognition functions of XPC-hHR23B and XPA-RPA in nucleotide excision repair.

It has been reported that 80-90% of human cancers may result, in part, from DNA damage. Cell survival depends critically on the stability of our DNA and exquisitely sensitive DNA repair mechanisms have developed as a result. In humans, nucleotide excision repair (NER) protects the DNA against the mutagenic effects of carcinogens and ultraviolet (UV) radiation from sun exposure. By preventing mutations from forming in the DNA, the repair machinery ultimately protects us from developing cancers. DNA damage recognition is the rate-limiting step in repair, and although many details of NER have been elucidated, the mechanisms by which DNA damage is recognized remain to be fully determined. Two primary protein complexes have been proposed as the damaged DNA recognition factor in NER: xeroderma pigmentosum protein A-replication protein A (XPA-RPA) and xeroderma pigmentosum protein C-human homolog of RAD23B (XPC-hHR23B). Here we compare the evidence that supports damage detection by these protein complexes and propose a model for DNA damage recognition in NER based on the current understanding of the roles these proteins may play in the processing of DNA lesions.

DNA↗
PubMed1994

Sequence-specific recognition of DNA by phenanthrenequinone diimine complexes of rhodium(III): importance of steric and van der Waals interactions.

The importance of steric and van der Waals interactions in the sequence-specific recognition of DNA by [Rh(phi)]3+ complexes has been explored through the synthesis and application of a series of Rh(phi)3+ (phi: 9,10-phenanthrenequinone diimine) derivatives. [Rh(phi)]3+ complexes intercalate in the major groove of DNA via the phi ligand and promote strand scission in the presence of UV light. The complexes reported here are derivatives of the parent molecules [Rh(phi)2bpy]3+ and [Rh(bpy)2phi]3+ (bpy: 2,2'-bipyridyl). The [Rh(phi)]3+ complexes have comparable photoefficiencies; therefore, their different photocleavage patterns on 32P-end-labeled DNA fragments reflect their unique sequence-specific recognition characteristics. The shapes of the [Rh(phi)]3+ complexes are found to govern DNA recognition and reaction. Importantly and generally, the more sterically bulky complexes, containing methyl or phenyl groups on the ancillary ligands, cleave DNA at a subset of sequences recognized by their parent molecules. [Rh-(diphenylbpy)2phi]3+ specifically targets the site 5'-CTCTAGAG-3'. Furthermore, chiral discrimination in site selectivity is observed; the different isomers target different sites. delta- and lambda-[Rh(5,5'-dimethylbpy)2phi]3+ cleave specifically at sites that are defined by the consensus sequences 5'-C-T-N-G-3' and 5'-A-C/G-T-C/G-3', respectively. The sequence selectivities may be understood on the basis of both negative steric clashes and positive van der Waals interactions between methyl groups on the metal complex and thymine methyl groups in the DNA major groove.

Base Sequence↗
PubMed2000

Reprogrammable recognition codes in bicoid homeodomain-DNA interaction.

We describe experiments to determine how the homeodomain of the Drosophila morphogenetic protein Bicoid recognizes different types of DNA sequences found in natural enhancers. Our chemical footprint analyses reveal that the Bicoid homeodomain makes both shared and distinct contacts with a consensus site A1 (TAATCC) and a nonconsensus site X1 (TAAGCT). In particular, the guanine of X1 at position 4 (TAAGCT) is protected by Bicoid homeodomain. We provide further evidence suggesting that the unique arginine at position 54 (Arg 54) of the Bicoid homeodomain enables the protein to recognize X1 by specifically interacting with this position 4 guanine. We also describe experiments to analyze the contribution of artificially introduced Arg 54 to DNA recognition by other Bicoid-related homeodomains, including that from the human disease protein Pitx2. Our experiments demonstrate that the role of Arg 54 varies depending on the exact homeodomain framework and DNA sequences. Together, our results suggest that Bicoid and its related homeodomains utilize distinct recognition codes to interact with different DNA sequences, underscoring the need to study DNA recognition by Bicoid-class homeodomains in an individualized manner.

Amino Acid Sequence↗
PubMed1992

Does cruciform DNA provide a recognition signal for DNA-topoisomerase II?

Topoisomerase II displays higher affinity for supercoiled DNA compared to the same relaxed DNA. Moreover, cruciform structures are formed in topologically constrained DNA. Here we report that, using S1 nuclease experiments on supercoiled DNA, hairpin structures are located close to numerous topoisomerase II cleavage sites on the BPV I genome. Therefore, DNA secondary structure may play a role in the recognition mechanism of DNA by topoisomerase II.

Base Sequence↗
PubMed2005

Conformation of DNA modified by monofunctional Ru(II) arene complexes: recognition by DNA binding proteins and repair. Relationship to cytotoxicity.

We analyzed DNA duplexes modified at central guanine residues by monofunctional Ru(II) arene complexes [(eta(6)-arene)Ru(II)(en)(Cl)](+) (arene = tetrahydroanthracene or p-cymene, Ru-THA or Ru-CYM, respectively). These two complexes were chosen as representatives of two different classes of Ru(II) arene compounds for which initial studies revealed different binding modes: one that may involve DNA intercalation (tricyclic-ring Ru-THA) and the other (mono-ring Ru-CYM) that may not. Ru-THA is approximately 20 times more toxic to cancer cells than Ru-CYM. The adducts of Ru-THA and Ru-CYM have contrasting effects on the conformation, thermodynamic stability, and polymerization of DNA in vitro. In addition, the adducts of Ru-CYM are removed from DNA more efficiently than those of Ru-THA. Interestingly, the mammalian nucleotide excision repair system has low efficiency for excision of ruthenium adducts compared to cisplatin intrastrand crosslinks.

Base Sequence↗
PubMed2005

Synthesis of C-nucleosides designed to participate in triplex formation with native DNA: specific recognition of an A:T base pair in DNA.

[structure: see text] We have previously described a system of 2-aminoquinoline- and 2-aminoquinazoline-based C-deoxynucleosides (TRIPsides) that are designed to be incorporated into oligomers that can specifically bind in the major groove via Hoogsteen base pairing to any sequence of native DNA. The four TRIPsides are termed antiGC, antiCG, antiTA, and antiAT with respect to the Watson-Crick base pair targets that they bind. The first three TRIPsides have been prepared, characterized, and shown to form stable and sequence-specific triplexes. In the present study, we describe the preparation of two molecules, 2-amino-4-(2'-deoxy-beta-D-ribofuranosyl)quinazoline (7) and 2-amino-6-fluoro-4-(2'-deoxy-beta-D-ribofuranosyl)quinoline (14), that can serve as the remaining antiAT TRIPside. The phosphoramidites of 7 and 14 were prepared, but only the latter was successfully incorporated into DNA oligomers. It is demonstrated using UV-visible melting experiments that 14 forms sequence-specific intramolecular triplets with A:T base pairs at physiological pH.

Adenine↗
PubMed2002

Sequence-specific recognition of DNA by hydrophobic, alanine-rich mutants of the basic region/leucine zipper motif investigated by fluorescence anisotropy.

We generated minimalist proteins capable of sequence-specific, high-affinity binding of DNA to probe how proteins are used and can be used to recognize DNA. In order to quantify binding affinities and specificities in our protein-DNA system, we used fluorescence anisotropy to measure in situ the thermodynamics of binding of alanine-rich mutants of the GCN4 basic region/leucine zipper (bZIP) domain to DNA duplexes containing target sites AP-1 (5'-TGACTCA-3') or ATF/CREB (5'-TGACGTCA-3'). We simplified the alpha-helical bZIP molecular recognition scaffold by alanine substitution: 4A, 11A, and 18A contain four, eleven, and eighteen alanine mutations in their DNA-binding basic regions, respectively. DNase I footprinting analysis demonstrates that all bZIP mutants retain the sequence-specific DNA-binding function of native GCN4 bZIP. Titration of fluorescein-labeled oligonucleotide duplexes with increasing amounts of protein yielded low nanomolar dissociation constants for all bZIP mutants in complex with the AP-1 and ATF/CREB sites: binding to the nonspecific control duplex was > 1000-fold weaker. Remarkably, the most heavily mutated protein 18A, containing 24 alanines in its 27-residue basic region, still binds AP-1 and ATF/CREB with dissociation constants of 15 and 7.8 nM, respectively. Similarly, wild-type bZIP binds these sites with K(d) values of 9.1 and 14 nM. 11A also displays low nanomolar dissociation constants for AP-1 and ATF/CREB, while 4A binds these sites with approximately 10-fold weaker K(d) values. Thus, both DNA-binding specificity and affinity are maintained in all our bZIP derivatives. This Ala-rich scaffold may be useful in design and synthesis of small alpha-helical proteins with desired DNA-recognition properties capable of serving as therapeutics targeting transcription.

Alanine↗
PubMed1995

Mutation of the Oct-1 POU-specific recognition helix leads to altered DNA binding and influences enhancement of adenovirus DNA replication.

To assess which residues of Oct-1 POU-specific (POUs) are important for DNA recognition and stimulation of adenovirus DNA replication we have mutated 10 residues of the POUs helix-turn-helix motif implicated in DNA contact. Seven of these turned out to have reduced DNA binding affinity. Of these, three alanine substituted proteins were found to have a changed specificity using a binding site selection procedure. Mutation of the first residue in the recognition helix, Gln44, to alanine led to a loss of specificity for the first two bases, TA, of the wild-type recognition site TATGC(A/T)AAT. Instead of the A, a T was selected, suggesting a new contact and a novel specificity. A change in specificity was also observed for the T45A mutant, which could bind to TATAC(A/T)AAT, a site hardly recognized by the wild-type protein. Mutation of residue Arg49 led to a relaxed specificity for three consecutive bases, TGC. This residue, which is critical for high affinity binding, is absent from the structurally homologous lambdoid helix-turn-helix motifs. Employing a reconstituted system all but two mutants could stimulate adenovirus DNA replication upon saturation. Mutation of residues Gln27 and Arg49 impairs the ability of the Oct-1 POU domain protein to enhance replication, with a concomitant loss of DNA contacts. Since the POU domain binds the precursor terminal protein-DNA polymerase complex and guides it to the origin, lack of stimulation may be caused by incorrect targetting of the DNA polymerase due to loss of specificity.

Adenoviridae↗
PubMed2001

DNA damage recognition and repair pathway coordination revealed by the structural biochemistry of DNA repair enzymes.

Cells have evolved distinct mechanisms for both preventing and removing mutagenic and lethal DNA damage. Structural and biochemical characterization of key enzymes that function in DNA repair pathways are illuminating the biological and chemical mechanisms that govern initial lesion detection, recognition, and excision repair of damaged DNA. These results are beginning to reveal a higher level of DNA repair coordination that ensures the faithful repair of damaged DNA. Enzyme-induced DNA distortions allow for the specific recognition of distinct extrahelical lesions, as well as tight binding to cleaved products, which has implications for the ordered transfer of unstable DNA repair intermediates between enzymes during base excision repair.

Alkylation↗
PubMed2001

Crystal structure of a ternary SAP-1/SRF/c-fos SRE DNA complex.

Combinatorial DNA binding by proteins for promoter-specific gene activation is a common mode of DNA regulation in eukaryotic organisms, and occurs at the promoter of the c-fos proto-oncogene. The c-fos promoter contains a serum response element (SRE) that mediates ternary complex formation with the Ets proteins SAP-1 or Elk-1 and the MADS-box protein, serum response factor (SRF). Here, we report the crystal structure of a ternary SAP-1/SRF/c-fos SRE DNA complex containing the minimal DNA-binding domains of each protein. The structure of the complex reveals that the SAP-1 monomer and SRF dimer are bound on opposite faces of the DNA, and that the DNA recognition helix of SAP-1 makes direct contact with the DNA recognition helix of one of the two SRF subunits. These interactions facilitate an 82 degrees DNA bend around SRF and a modulation of protein-DNA contacts by each protein when compared to each of the binary DNA complexes. A comparison with a recently determined complex containing SRF, an idealized DNA site, and a SAP-1 fragment containing a SRF-interacting B-box region, shows a similar overall architecture but also shows important differences. Specifically, the comparison suggests that the B-box region of the Ets protein does not significantly influence DNA recognition by either of the proteins, and that the sequence of the DNA target effects the way in which the two proteins cooperate for DNA recognition. These studies have implications for how DNA-bound SRF may modulate the DNA-binding properties of other Ets proteins such as Elk-1, and for how other Ets proteins may modulate the DNA-binding properties of other DNA-bound accessory factors to facilitate promoter-specific transcriptional responses.

Amino Acid Sequence↗
PubMed2004

Control of Hap1-DNA site recognition through the interplay of multiple distinct intermolecular interactions.

Hap1 belongs to the Zn(2)Cys(6) zinc binuclear cluster family of transcription factors that typically bind as dimers to symmetric DNA sites containing two CGG triplets separated by spacer DNA. The cluster domain binds CGG while an adjoining C-terminal linker and dimerization helix specifies the length of spacer DNA recognized. Hap1 is unusual in binding a direct repeat of CGG triplets, in contacting a TA in the spacer DNA, and in making direct dimer contacts between its cluster domains. Binding of Hap1 fragments to different DNA sites was tested to determine how these interactions control Hap1-DNA recognition. The spacer TA contacts were found to facilitate monomer binding of Hap1 to a single CGG. When the spacer-binding residues were deleted, binding was still specific for the direct repeat but was much weaker and appeared to require dimerization. When the dimerization helix and all subsequent C-terminal residues were deleted, the remaining linker, cluster domain, and spacer-binding residues still dimerized on DNA. The energy of this dimerization was comparable to that of the Hap1-spacer TA interaction. Moving the TA from the spacer to a position following the second CGG maintained Hap1 monomer binding but greatly weakened dimerization. This suggested that binding a TA after the second CGG triplet required a geometry that impaired dimerization with a Hap1 molecule on the first CGG. The geometric restraints for optimal TA binding and dimerization thus drive Hap1 selectivity for CGG direct repeat sites that contain an asymmetrically positioned spacer TA following the first CGG triplet.

Carrier Proteins↗
PubMed1999

Synergy of importin alpha recognition and DNA binding by the yeast transcriptional activator GAL4.

The N-terminus of the yeast transcriptional activator GAL4 contains partially overlapping nuclear targeting and DNA binding functions. We have previously shown that GAL4 is recognised with high affinity by importin beta and not by the conventional nuclear localisation sequence binding importin alpha subunit of the importin alpha/beta heterodimer. The present study uses ELISA-based binding and electrophoretic mobility shift assays to show that recognition of GAL4 by importin alpha can occur, but only when GAL4 is bound to its specific DNA recognition sequence. Intriguingly, binding by importin alpha enhances DNA binding on the part of GAL4, implying a synergistic co-operation between these two functions. The results implicate a possible role for importin alpha in the nucleus additional to its established role in nuclear transport, as well as having implications for the use of GAL4 as a DNA carrier in gene therapy applications.

Animals↗
PubMed2003

Chimeric recombinases with designed DNA sequence recognition.

Site-specific recombination typically occurs only between DNA sequences that have co-evolved with a natural recombinase enzyme to optimize sequence recognition, catalytic efficiency, and regulation. Here, we show that the sequence recognition and the catalysis functions of a recombinase can be specified by unrelated protein domains. We describe chimeric recombinases with a catalytic domain from an activated multiple mutant of the bacterial enzyme Tn3 resolvase, fused to a DNA recognition domain from the mouse transcription factor Zif268. These proteins catalyze efficient recombination specifically at synthetic target sites recognized by two Zif268 domains. Our results demonstrate the functional autonomy of the resolvase catalytic domain and open the way to creating "custom-built" recombinases that act at chosen natural target sequences.

Amino Acid Sequence↗
PubMed2001

Restriction enzyme BsoBI-DNA complex: a tunnel for recognition of degenerate DNA sequences and potential histidine catalysis.

BACKGROUND: Restriction endonucleases form a diverse family of proteins with substantial variation in sequence, structure, and interaction with recognition site DNA. BsoBI is a thermophilic restriction endonuclease that exhibits both base-specific and degenerate recognition within the sequence CPyCGPuG. RESULTS: The structure of BsoBI complexed to cognate DNA has been determined to 1.7 A resolution, revealing several unprecedented features. Each BsoBI monomer is formed by inserting a helical domain into an expanded EcoRI-type catalytic domain. DNA is completely encircled by a BsoBI dimer. Recognition sequence DNA lies within a 20 A long tunnel of protein that excludes bulk solvent. Interactions with the specific bases are made in both grooves through direct and water-mediated hydrogen bonding. Interaction with the degenerate position is mediated by a purine-specific hydrogen bond to N7, ensuring specificity, and water-mediated H bonding to the purine N6/O6 and pyrimidine N4/O4, allowing degeneracy. In addition to the conserved active site residues of the DX(n)(E/D)ZK restriction enzyme motif, His253 is positioned to act as a general base. CONCLUSIONS: A catalytic mechanism employing His253 and two metal ions is proposed. If confirmed, this would be the first example of histidine-mediated catalysis in a restriction endonuclease. The structure also provides two novel examples of the role of water in protein-DNA interaction. Degenerate recognition may be mediated by employing water as a hydrogen bond donor or acceptor. The structure of DNA in the tunnel may also be influenced by the absence of bulk solvent.

Catalysis↗