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At least 289 records · Page 16Linked to original sources
PubMed2006

Characterization of the binding of distamycin A to damaged DNA: a comparison with the DNA recognition of the human DDB protein.

Distamycin A binds to DNA containing the (6-4) photoproduct, a major UV lesion that is recognized by the damaged DNA-binding (DDB) protein in human cells. We analyzed the binding properties of distamycin A and compared the results with those of the DDB protein. Structural change of the DNA duplex was not observed for distamycin A in two types of experiments, whereas the protein induced a large bending of the helix. Although the substrate specificity was different between the drug and the protein, thymine glycol was recognized by both of them, and inhibition of the DDB protein binding to the (6-4) photoproduct-containing DNA by distamycin A was tested.

Binding Sites↗
PubMed1999

Structural basis of DNA recognition by the heterodimeric cell cycle transcription factor E2F-DP.

The E2F and DP protein families form heterodimeric transcription factors that play a central role in the expression of cell cycle-regulated genes. The crystal structure of an E2F4-DP2-DNA complex shows that the DNA-binding domains of the E2F and DP proteins both have a fold related to the winged-helix DNA-binding motif. Recognition of the central c/gGCGCg/c sequence of the consensus DNA-binding site is symmetric, and amino acids that contact these bases are conserved among all known E2F and DP proteins. The asymmetry in the extended binding site TTTc/gGCGCc/g is associated with an amino-terminal extension of E2F4, in which an arginine binds in the minor groove near the TTT stretch. This arginine is invariant among E2Fs but not present in DPs. E2F4 and DP2 interact through an extensive protein-protein interface, and structural features of this interface suggest it contributes to the preference for heterodimers over homodimers in DNA binding.

Amino Acid Sequence↗
PubMed1992

EcoRV restriction endonuclease: communication between catalytic metal ions and DNA recognition.

In the absence of magnesium ions, the EcoRV restriction endonuclease binds all DNA sequences with equal affinity but cannot cleave DNA. In the presence of Mg2+, the EcoRV endonuclease cleaves DNA at one particular sequence, GATATC, at least a million times more readily than any other sequence. To elucidate the role of the metal ion, the reactions of the EcoRV restriction enzyme were studied in the presence of MnCl2 instead of MgCl2. The reaction at the EcoRV recognition site was slower with Mn2+. This was caused partly by reduced rates for phosphodiester hydrolysis but also by the translocation of the enzyme along the DNA after cleaving it in one strand. In contrast, alternative sites that differ from the recognition site by one base pair were cleaved faster in the presence of Mn2+ relative to Mg2+. When located at an alternative site on the DNA, the EcoRV enzyme bound Mn2+ ions readily but had a very low affinity for Mg2+. The EcoRV nuclease is thus restrained from cleaving DNA at alternate sites in the presence of Mg2+, but the restraint fails to operate with Mn2+. A discrimination factor, which measures the ratio of the activity of the EcoRV nuclease at its recognition site over that at an alternative site, had values of 3 x 10(5) in MgCl2 and 6 in MnCl2.

Base Sequence↗
PubMed1994

MONTY: a Monte Carlo approach to protein-DNA recognition.

A Monte Carlo method is described for automated docking of proteins on DNA. The simulation program MONTY keeps the entire DNA and the protein backbone and core fixed while protein surface side-chains are allowed to rotate freely. The entire protein is rotated and translated by small random steps in order to find the best fit with the DNA. New configurations are accepted on basis of their Boltzmann probability. Protein-DNA interaction is represented by square well potentials for hydrogen bond and van der Waals interactions. The structure with the largest interaction energy encountered during the simulation is saved. The method is tested on complexes of the 434 Cro protein and its operator DNA where the protein is shifted up or down one or two base-pairs and is subsequently allowed to find back its native binding site. This protocol is performed for shifted complexes derived from the crystal structure, shifted complexes where the crystal structure DNA is replaced by standard B-DNA and shifted complexes where in addition the protein is replaced by protein from the uncomplexed crystal structure. In all three cases the six lowest energy structures correspond to complexes close to the native complex. The quality of sequence specific recognition diminishes, however, when the molecular surface complementarity between protein and DNA decreases.

Base Composition↗
PubMed1996

Raman spectroscopy of the Ff gene V protein and complexes with poly(dA): nonspecific DNA recognition and binding.

Raman spectra of crystals and solutions of the single-stranded DNA binding protein of bacteriophage Ff (gene V protein, gVp) and of solution complexes of gVp with single-stranded poly-(deoxyadenylic acid) [poly(dA)] reveal the following: (i) The gVp secondary and tertiary structures are similar in solution and in the crystal and are dominated by beta-sheet domains, in agreement with NMR and X-ray findings. (ii) Subunit conformation and side chain environments of gVp are virtually unchanged over a wide range of salt concentration (0 < [NaCl] < 100 mM); however, the solution conformation of poly(dA) exhibits sensitivity to added salt. The perturbed Raman markers indicate subtle changes in helix backbone geometry with accompanying small differences in base stacking as the concentration of NaCl is changed. (iii) In complexes with poly(dA), neither the conformation of gVp nor its side chain environments are altered significantly in comparison to the free protein. This is the case at both high salt (nucleotide-to-subunit binding stoichiometry n = 4) and low salt (n = 3). (iv) The Raman signature of poly(dA) undergoes small perturbations upon gVp binding, indicative of small changes in base stacking and phosphodiester backbone conformation. The present results show that the different stoichiometric binding modes of gVp to poly(dA) are accomplished without significant changes in gVp subunit structure and with only modest changes in the single-stranded poly(dA) ligand. This contrasts sharply with sequence-specific double-stranded DNA binding proteins, such as the phage lambda and D108 repressors, which undergo substantial structural changes upon DNA binding, and which also alter more dramatically the Raman fingerprints of their DNA target sites. Thus, nonspecific and specific nucleic acid recognition modes are distinguishable by Raman spectroscopy. The Raman signature of gVp also allows examination of hydrogen bonding interactions of unique side chains within the hydrophobic core (cysteine 33) and at the binding interface (tyrosine 41). These are discussed in relation to the recently published gVp crystal structure.

Amino Acid Sequence↗
PubMed1991

Human immunodeficiency virus integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage.

Retroviral integration requires cis-acting sequences at the termini of linear double-stranded viral DNA and a product of the retroviral pol gene, the integrase protein (IN). IN is required and sufficient for generation of recessed 3' termini of the viral DNA (the first step in proviral integration) and for integration of the recessed DNA species in vitro. Human immunodeficiency virus type 1 (HIV-1) IN, expressed in Escherichia coli, was purified to near homogeneity. The substrate sequence requirements for specific cleavage and integration of retroviral DNA were studied in a physical assay, using purified IN and short duplex oligonucleotides that correspond to the termini of HIV DNA. A few point mutations around the IN cleavage site substantially reduced cleavage; most other mutations did not have a drastic effect, suggesting that the sequence requirements are limited. The terminal 15 bp of the retroviral DNA were demonstrated to be sufficient for recognition by IN. Efficient specific cutting of the retroviral DNA by IN required that the cleavage site, the phosphodiester bond at the 3' side of a conserved CA-3' dinucleotide, be located two nucleotides away from the end of the viral DNA; however, low-efficiency cutting was observed when the cleavage site was located one, three, four, or five nucleotides away from the terminus of the double-stranded viral DNA. Increased cleavage by IN was detected when the nucleotides 3' of the CA-3' dinucleotide were present as single-stranded DNA. IN was found to have a strong preference for promoting integration into double-stranded rather than single-stranded DNA.

Base Sequence↗
PubMed1997

Distinct HOX N-terminal arm residues are responsible for specificity of DNA recognition by HOX monomers and HOX.PBX heterodimers.

Dimerization with extradenticle or PBX homeoproteins dramatically improves DNA binding by HOX transcription factors, indicating that recognition by such complexes is important for HOX specificity. For HOX monomeric binding, a major determinant of specificity is the flexible N-terminal arm. It makes base-specific contacts via the minor groove, including one to the 1st position of a 5'-TNAT-3' core by a conserved arginine (Arg-5). Here we show that Arg-5 also contributes to the stability of HOX.PBX complexes, apparently by forming the same DNA contact. We further show that heterodimers of PBX with HOXA1 or HOXD4 proteins have different specificities at another position recognized by the N-terminal arm (the 2nd position in the TNAT core). Importantly, N-terminal arm residues 2 and 3, which distinguish the binding of HOXA1 and HOXD4 monomers, play no role in the specificity of their complexes with PBX. In addition, HOXD9 and HOXD10, which are capable of binding both TTAT and TAAT sites as monomers, can cooperate with PBX1A only on a TTAT site. These data suggest that some DNA contacts made by the N-terminal arm are altered by interaction with PBX.

Amino Acid Sequence↗
PubMed1996

A noncovalent binding-translocation mechanism for site-specific CC-1065-DNA recognition.

The molecular strategy by which small organic compounds recognise specific DNA sequences is of primary importance for rational drug design. CC-1065 is a potent alkylating agent that binds covalently to N3 of adenine and lies in the minor groove of double-stranded DNA. Its reaction with DNA occurs in a site-specific manner, with a preference for A. T-rich nucleotide sequences. In the present study, we developed a drug translocation assay to investigate the mechanism underlying this sequence selectivity. After exposure of plasmid DNA to saturating amounts of CC-1065, we observed that nearly 70% of plasmid-bound CC-1065 molecules formed stable, but noncovalent, complexes with DNA. These noncovalently bound drug molecules resisted purification by ethanol precipitation, dialysis, and sucrose gradient centrifugation, but retained the ability to translocate to DNA fragments containing a single high-affinity site for alkylation. This combination of non-covalent binding interactions and drug translocation provides a mechanism by which CC-1065 may locate specific alkylation sites in DNA.

Alkylation↗
PubMed2003

Cooperative dimerization of a heterocyclic diamidine determines sequence-specific DNA recognition.

In the course of a program aimed at discovering novel DNA-targeted antiparasitic drugs, the phenylfuran-benzimidazole unfused aromatic dication DB293 was identified as the first diamidine capable of forming stacked dimers in the DNA minor groove of GC-containing sequences. Its preferred binding sequence encompasses the tetranucleotide 5'-ATGA.5'-TCAT to which DB293 binds tightly with a strong positive cooperativity. Here we have investigated the influence of the DNA sequence on drug binding using two complementary technical approaches: surface plasmon resonance and DNase I footprinting. The central dinucleotide of the primary ATGA motif was systematically varied to represent all of the eight possible combinations (AXGA and ATYA, where X or Y = A, T, G, or C). Binding affinities for each site were precisely measured by SPR, and the extent of cooperative drug binding was also determined. The sequence recognition process was found to be extremely dependent on the nature of the central dinucleotide pair. Modification of the central TG step decreases binding affinity by a factor varying from 2 to over 500 depending on the base substitution. However, the diminished binding affinity does not affect the unique binding mode. In nearly all cases, the SPR titrations revealed a positive cooperativity in complex formation which reflects the ease of the dication to form stacked dimeric motifs in the DNA minor groove. DNase I footprinting served to identify additional binding sites for DB293 in the context of long DNA sequences offering a large variety of randomly distributed or specifically designed sites. The ATGA motif provided the best receptor for the drug, but lower affinity sequences were also identified. The design of two DNA fragments composed of various targeted tetranucleotide binding sites separated by an "insulator" (nonbinding) sequence allowed us to delineate further the influence of DNA sequence on drug binding and to identify a novel high-affinity site: 5'-ACAA.5'-TTGT. Collectively, the SPR and footprinting results show that the consensus sequence 5'-(A/T)-TG-(A/T) represents the optimal site for cooperative dimerization of the heterocyclic diamidine DB293.

Base Sequence↗
PubMed1994

Two functionally distinct half sites in the DNA-recognition sequence of the Myb oncoprotein.

The oncoprotein Myb is a sequence-specific DNA-binding protein with a pivotal function in the development and proliferation of hematopoietic precursor cells. A minimal DNA-binding domain composed of two tryptophan-rich repeats R2 and R3 is responsible for sequence recognition. Based on model building and mutational analysis, Myb was proposed to recognise its target through a double helix-turn-helix (HTH)-related motif using two recognition helices, one in R2 and one in R3. We found, by mutational analysis, that the DNA-binding site for c-Myb is functionally bipartite. While the first half site is dominant and absolutely required for binding, the second half site is only modulatory and mainly affects the half life of the complex. This bipartite nature of the binding site parallels the proposed bipartite structure of R2R3 with two HTH-related domains. Analysis of the DNA-binding site of R2R3 by missing-base interference-footprint analysis showed that the protein interacted with a 9-bp region. The same was found with a larger protein containing all three repeats. The effect of adding R1 was mainly to stabilise the complex. The borders of the complex, as revealed by exonuclease III footprinting, did not change due to the presence of R1. However, both borders became more refractory to the nuclease when R1 was present, but with a difference that suggested a specific orientation of the repeat domains relative to the DNA-binding site. We propose that the first half site is recognised by R3, while the second modulatory half site interacts with the R2 repeat.

Amino Acid Sequence↗
PubMed1993

Macroevolution by transposition: drastic modification of DNA recognition by a type I restriction enzyme following Tn5 transposition.

We have characterized a novel mutant of EcoDXXI, a type IC DNA restriction and modification (R-M) system, in which the specificity has been altered due to a Tn5 insertion into the middle of hsdS, the gene which encodes the polypeptide that confers DNA sequence specificity to both the restriction and the modification reactions. Like other type I enzymes, the wild type EcoDXXI recognizes a sequence composed of two asymmetrical half sites separated by a spacer region: TCA(N7)RTTC. Purification of the EcoDXXI mutant methylase and subsequent in vitro DNA methylation assays identified the mutant recognition sequence as an interrupted palindrome, TCA(N8)TGA, in which the 5' half site of the wild type site is repeated in inverse orientation. The additional base pair in the non-specific spacer of the mutant recognition sequence maintains the proper spacing between the two methylatable adenine groups. Sequencing of both the wild type and mutant EcoDXXI hsdS genes showed that the Tn5 insertion occurred at nucleotide 673 of the 1221 bp gene. This effectively deletes the entire carboxyl-terminal DNA binding domain which recognizes the 3' half of the EcoDXXI binding site. The truncated hsdS gene still encodes both the amino-terminal DNA binding domain and the conserved repeated sequence that defines the length of the recognition site spacer region. We propose that the EcoDXXI mutant methylase utilizes two truncated hsdS subunits to recognize its binding site. The implications of this finding in terms of subunit interactions and the malleability of the type I R-M systems will be discussed.

Base Sequence↗
PubMed1993

Inhibition of UV-damaged DNA recognition activity in HeLa cells by calcium ionophore A23187: potential involvement of cytosolic proteins.

A UV-damage recognition protein (UVDRP) was detected from HeLa nuclear extracts using an in vitro DNA-binding assay. Treatment of cells with a Ca2+ ionophore (A23187) caused a dramatic inhibition of the UVDRP binding activity from nuclear extracts. In the absence of EDTA, addition of cytosolic extracts to the binding reaction effectively blocked the UVDRP binding activity, associated with an increased nuclease activity. However, pretreatment of cytosolic extracts with proteinase destroyed a majority of the inhibitory effect on UVDRP binding activity. Using extracts from A23187-treated cells, a similar conclusion was drawn except that the inhibitory effect of cytosolic extracts on UVDRP binding was completely diminished by proteinase K. In addition, inclusion of the cytosolic extracts in the binding reaction did not alter the UVDRP binding dissociation pattern by specific competitors. These results suggest the potential involvement of cytosolic proteins, probably through a "all-or-none" action mechanism, in the recognition of UV-damaged DNA of HeLa cells.

Calcimycin↗
PubMed1998

Functionalized Rhodium Intercalators for DNA Recognition.

A series of rhodium complexes containing the phenanthrenequinone diimine (phi) ligand have been prepared which bind DNA by intercalation and, upon photoactivation, promote DNA strand breaks. In this series, the ancillary, nonintercalating bipyridyl or phenanthroline ligands have been functionalized to yield complexes containing guanidinium, amido, or amino groups arranged with defined stereochemistry for site-specific interaction with the DNA bases. Lambda-1-[Rh(MGP)(2)phi](5+) (MGP = 4-(guanidylmethyl)-1,10-phenanthroline) site-specifically targets the 6-base pair sequence 5'-CATATG-3' with a binding affinity of 1 (+/-0.5) x 10(8) M(-)(1) while Delta-1-[Rh(MGP)(2)phi](5+) displays an affinity of 5 (+/-2) x 10(7) M(-)(1) for 5'-CATCTG-3'. Even though these two isomers target sites which differ by only a single base, binding is highly enantioselective. The specificity is derived chiefly from interactions of the pendant guanidinium groups with the DNA bases. For the racemates of 1-[Rh(GEB)(2)phi](5+) (GEB = (4-(2-guanidylethyl)-4'-methyl-2,2'-bipyridine) and 1-[Rh(GPB)(2)phi](5+) (GPB = (4-(2-guanidylpropyl)-4'-methyl-2,2'-bipyridine), photocleavage patterns also show the strongest site of photocleavage as 5'-CATCTG-3', the target site for Delta-1-[Rh(MGP)(2)phi](5+). Moreover, consistent with the dominance of the guanidinium groups in establishing specificity, significantly enhanced photocleavage is evident for the 1-positional isomer of these complexes, where the guanidinium moieties are directed toward the DNA (above and below the phi ligand) compared to the 2-isomer, in which the guanidinium groups are directed away from the DNA. In contrast to Lambda-1-[Rh(MGP)(2)phi](5+), Lambda-1-[Rh(GEB)(2)phi](5+) shows little cleavage at 5'-CATATG-3'; this sensitivity to linker length likely depends on the mode of recognition of 5'-CATATG-3' involving sequence-dependent unwinding of the DNA site. Analogous site-specificity or isomer-specificity is not evident with the complexes which contain pendant amido or amino functionalities. Instead these complexes appear to resemble the parent, unfunctionalized [Rh(phen)(2)phi](3+) with respect to recognition. Pendant guanidinium functionalities appear to be particularly advantageous in the construction of small molecules which bind DNA with site-specificity.

Journal Article↗
PubMed1998

Differential DNA recognition by the enantiomers of 1-Rh(MGP)2 phi: a combination of shape selection and direct readout.

The enantiomers of the symmetric metallointercalator complex 1-Rh(MGP)2phi5+ [MGP = 4-(guanidylmethyl)-1,10-phenanthroline; phi = phenanthrenequinone diimine] bound to DNA decamer duplexes containing their respective 6 bp recognition sequences have been investigated using 1H NMR. Shape selection due to the chirality of the metal center and hydrogen-bonding contacts of ancillary guanidinium groups to 3'-G N7 atoms define the recognition by complexes which bind by intercalation to duplex DNA. The titration of Lambda-Rh into the self-complementary decamer containing the recognition sequence (5'-GACATATGTC-3', L1) resulted in one symmetric bound conformation observed in the 1H NMR spectrum, indicating that the DNA duplex retains its symmetry in the presence of the metal complex. Upfield chemical shifts of duplex imino protons and the disruption of the NOE base-sugar contacts defined the central T5-A6 intercalation site. The downfield shift of the G8 imino proton supports the conclusion that the pendant guanidinium arms make simultaneous H-bonding contacts to the N7 atoms of 3'-G8 bases on either side of the site. A variable-temperature study of a partially titrated sample (2:3 Lambda-Rh/L1) showed the exchange rate (kobs) at 298 K to be 68 s-1 and the activation barrier to exchange (DeltaG of association) to be 2.7 kcal/mol, a value comparable to the stacking energy of one base step. The results presented coupled with biochemical data are therefore consistent with binding models in which Lambda-1-Rh(MGP)2phi5+ (Lambda-Rh) traps the recognition site 5'-CATATG-3' in an unwound state, permitting intercalation centrally and hydrogen bonding to guanines at the first and sixth base pair positions. The data suggest a different model of binding and recognition by Delta-Rh. The titration of Delta-Rh into a DNA decamer containing the 6 bp recognition site (D1, 5'-CGCATCTGAC-3'; D2, 5'-GTCAGATGCG-3') resulted in two, distinct conformers, in slow exchange on the NMR time scale. The rate of exchange between the two conformers (kobs) at 298 K is 37 s-1, most likely due to partial dissociation between binding modes. The slower rate relative to Lambda-Rh association reflects the relative rigidity of the D1 and/or D2 sequence in comparison to L1. NOE cross-peaks between the intercalating phi ligand and protons of T5-C6, as well as the upfield shifts observed for imino protons at this step, serve to define the central T5-C6 step as the single site of intercalation. The downfield shift of the 3'-G imino protons indicates the complex makes hydrogen bond contacts with these bases. The complex, which is too small to span a 6 bp B-form DNA sequence, nonetheless makes major groove contacts with 3'-G bases to either side of the site. Notably, both 3'-guanine bases are necessary to impart site specificity and slow dissociation kinetics with the 5'-CATCTG-3' site, as evidenced by the extremely exchange-broadened two-dimensional NOESY spectra of Delta-Rh bound to modified duplexes containing N7-deazaguanine at either G8 or G18; the loss of one major groove contact completely abolishes specificity for 5'-CATCTG-3'. DNA chemical shifts upon binding and intermolecular NOE contacts therefore support a model in which Delta-Rh intercalates in one of two canted binding conformations. Within this model, each intercalation mode allows one guanidinium-guanine hydrogen bond at a time, while bringing the other arm close to the phosphate backbone.

Binding Sites↗
PubMed2005

Differential DNA recognition and cleavage by EcoRI dependent on the dynamic equilibrium between the two forms of the malondialdehyde-deoxyguanosine adduct.

DNA damage may alter the outcome of protein-nucleic acid interactions. The malondialdehyde-deoxyguanosine adduct, 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10-(3H)-one (M(1)dG), miscodes in vivo and in vitro. M(1)dG is an exocyclic adduct that undergoes ring-opening in duplex DNA to form the acyclic adduct, N(2)-(3-oxo-1-propenyl)-deoxyguanosine (N(2)-OPdG). These two adducts have different effects on DNA polymerase bypass and may affect other DNA processing enzymes. We employed the EcoRI restriction endonuclease as a model for the interaction of DNA binding proteins with adducted DNA substrates. The presence of M(1)dG in the EcoRI recognition sequence impaired the ability of the enzyme to cleave DNA, resulting in only 60% cleavage of the adducted strand and 75% cleavage of the complementary strand. Three adducts of similar structure to M(1)dG that are unable to ring-open were cleaved poorly, or not at all, by EcoRI. None of the adducts appeared to inactivate or sequester EcoRI. Additional studies with BssHII and PauI confirmed these results and demonstrated a positional effect of M(1)dG on cleavage efficiency. These data suggest dissimilar modes of protein-nucleic acid interactions based on differences in adduct structure. Comparison of the solution structures of DNA adducts and the crystal structure of EcoRI complexed to substrate suggest a model to explain the functional differences.

Base Sequence↗
PubMed2005

DNA polymerase clamp loaders and DNA recognition.

Clamp loaders are heteropentameric ATPase assemblies that load sliding clamps onto DNA and are critical for processive DNA replication. The DNA targets for clamp loading are double-stranded/single-stranded junctions with recessed 3' ends (primer-template junctions). Here, we briefly review the crystal structures of clamp loader complexes and the insights they have provided into the mechanism of the clamp loading process.

Adenosine Triphosphatases↗