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PubMed2000

Recognition of the DNA minor groove by pyrrole-imidazole polyamides: comparison of desmethyl- and N-methylpyrrole.

Polyamides consisting of N-methylpyrrole (Py), N-methylimidazole (Im), and N-methyl-3-hydroxypyrrole (Hp) are synthetic ligands that recognize predetermined DNA sequences with affinities and specificities comparable to many DNA-binding proteins. As derivatives of the natural products distamycin and netropsin, Py/Im/Hp polyamides have retained the N-methyl substituent, although structural studies of polyamide:DNA complexes have not revealed an obvious function for the N-methyl. In order to assess the role of the N-methyl moiety in polyamide:DNA recognition, a new monomer, desmethylpyrrole (Ds), where the N-methyl moiety has been replaced with hydrogen, was incorporated into an eight-ring hairpin polyamide by solid-phase synthesis. MPE footprinting, affinity cleavage, and quantitative DNase I footprinting revealed that replacement of each Py residue with Ds resulted in identical binding site size and orientation and similar binding affinity for the six-base-pair (bp) target DNA sequence. Remarkably, the Ds-containing polyamide exhibited an 8-fold loss in specificity for the match site versus a mismatched DNA site, relative to the all-Py parent. Polyamides with Ds exhibit increased water solubility, which may alter the cell membrane permeability properties of the polyamide. The addition of Ds to the repertoire of available monomers may prove useful as polyamides are applied to gene regulation in vivo. However, the benefits of Ds incorporation must be balanced with a potential loss in specificity.

Autoradiography↗
PubMed1985

Mutational studies with the trp repressor of Escherichia coli support the helix-turn-helix model of repressor recognition of operator DNA.

Several classes of trp repressor mutants were selected and analyzed in vivo. Mutants that produced repressors with either enhanced or reduced activity were obtained. One class of mutants produced inactive or slightly active repressors that were trans-dominant to the wild-type repressor. The amino acid substitutions in many of these repressors were clustered in a segment of the polypeptide that is homologous to the DNA recognition domain of the lambda cro repressor. A second functionally important region of the trp repressor was identified; this region could participate in L-tryptophan binding. Observations with trpR nonsense mutants suggest that the first 67 residues of the repressor polypeptide are sufficient for subunit association.

Amino Acid Sequence↗
PubMed1991

Recognition of unusual DNA structures by human DNA (cytosine-5)methyltransferase.

The symmetry of the responses of the human DNA (cytosine-5)methyltransferase to alternative placements of 5-methylcytosine in model oligodeoxynucleotide duplexes containing unusual structures has been examined. The results of these experiments more clearly define the DNA recognition specificity of the enzyme. A simple three-nucleotide recognition motif within the CG dinucleotide pair can be identified in each enzymatically methylated duplex. The data can be summarized by numbering the four nucleotides in the dinucleotide pair thus: 1 4/2 3. With reference to this numbering scheme, position 1 can be occupied by cytosine or 5-methylcytosine; position 2 can be occupied by guanosine or inosine; position 3, the site of enzymatic methylation, can be occupied only by cytosine; and position 4 can be occupied by guanosine, inosine, O6-methylguanosine, cytosine, adenosine, an abasic site, or the 3' hydroxyl group at the end of a gapped molecule. Replacing the guanosine normally found at position 4 with any of the moieties introduces unusual (non-Watson-Crick) pairing at position 3 and generally enhances methylation of the cytosine at that site. The exceptional facility of the enzyme in actively methylating unusual DNA structures suggests that the evolution of the DNA methyltransferase, and perhaps DNA methylation itself, may be linked to the biological occurrence of unusual DNA structures.

5-Methylcytosine↗
PubMed1987

Role of glutamic acid-181 in DNA-sequence recognition by the catabolite gene activator protein (CAP) of Escherichia coli: altered DNA-sequence-recognition properties of [Val181]CAP and [Leu181]CAP.

It has been proposed that Glu-181 of the catabolite gene activator protein (CAP) makes direct contact with certain base pairs of the specific DNA site. We have purified wild-type CAP and two substituted CAP variants, [Val181]CAP and [Leu181]CAP, and have assessed the DNA-sequence-recognition properties in vitro with respect to positions 5, 6, 7, 8, and 16 of the DNA site. The data indicate that [Val181]CAP and [Leu181]CAP fail to discriminate between the consensus DNA base pair and the three non-consensus-DNA base pairs at 2-fold-related positions 7 and 16 of the DNA site. In contrast, [Val181]CAP and [Leu181]CAP retain the ability to discriminate between different base pairs at positions 5 and 8 of the DNA site. We conclude that Glu-181 of CAP makes a direct contact with 2-fold-related positions 7 and 16 of the DNA site, as proposed previously based on in vivo results. We propose that upon replacement of Glu-181 by valine or leucine, this contact is eliminated and is replaced by no other functional contact. We estimate that the contact by Glu-181 with each position contributes -0.7 kcal/mol to the total CAP-DNA binding free energy.

Amino Acid Sequence↗
PubMed2003

DNA sequence recognition by bispyrazinonaphthalimides antitumor agents.

Bifunctional DNA intercalating agents have long attracted considerable attention as anticancer agents. One of the lead compounds in this category is the dimeric antitumor drug elinafide, composed of two tricyclic naphthalimide chromophores separated by an aminoalkyl linker chain optimally designed to permit bisintercalation of the drug into DNA. In an effort to optimize the DNA recognition capacity, different series of elinafide analogues have been prepared by extending the surface of the planar drug chromophore which is important for DNA sequence recognition. We report here a detailed investigation of the DNA sequence preference of three tetracyclic monomeric or dimeric pyrazinonaphthalimide derivatives. Melting temperature measurements and surface plasmon resonance (SPR) studies indicate that the dimerization of the tetracyclic planar chromophore considerably augments the affinity of the drug for DNA, polynucleotides, or hairpin oligonucleotides and promotes selective interaction with G.C sites. The (CH(2))(2)NH(CH(2))(3)NH(CH(2))(2) connector stabilizes the drug-DNA complexes. The methylation of the two nitrogen atoms of this linker chain reduces the binding affinity and increases the dissociation rates of the drug-DNA complexes by a factor of 10. DNase I footprinting experiments were used to investigate the sequence selectivity of the drugs, demonstrating highly preferential binding to G.C-rich sequences. It also served to select a high-affinity site encompassing the sequence 5'-GACGGCCAG which was then introduced into a biotin-labeled hairpin oligonucleotide to accurately measure the binding parameters by SPR. The affinity constant of the unmethylated dimer for this sequence is 500 times higher than that of the monomer compound and approximately 10 times higher than that of the methylated dimer. The DNA groove accessibility was also probed with three related oligonucleotides carrying G --> c(7)G, G --> I, and C --> M substitutions. The level of drug binding to the two hairpin oligonucleotides containing 7-deazaguanine (c(7)G) or 5-methylcytosine (M) residues is unchanged or only slightly reduced compared to that of the unmodified target. In contrast, incorporation of inosine (I) residues considerably decreases the extent of drug binding or even abolishes the interaction as is the case with the monomer. The pyrazinonaphthalimide derivatives are thus much more sensitive to the deletion of the exocyclic guanine 2-amino group exposed in the minor groove of the duplex than to the modification of the major groove elements. The complementary SPR footprinting methodology combining site selection and quantitative DNA affinity analysis constitutes a reliable method for dissecting the DNA sequence selectivity profile of reversible DNA binding small molecules.

Adenine↗
PubMed2004

A model for initial DNA lesion recognition by NER and MMR based on local conformational flexibility.

Initial recognition of DNA damage is the crucial but poorly understood first step in DNA repair by the human nucleotide excision repair(NER) and mismatch repair (MMR) systems. Failure by NER or MMR to recognize DNA damage threatens the genetic integrity of the organism and may play a role in carcinogenesis. Both NER and MMR recognize and repair a wide variety of structurally dissimilar lesions against the background of normal DNA. Previous studies have suggested that detection of thermodynamic destabilization of DNA caused by covalent damage and base mismatches is a potential mechanism by which repair pathways with broad specificity such as NER and MMR recognize their substrates. However, both NER and MMR respectively, repair a wide variety of stabilizing and destabilizing covalent DNA lesions and base pair mismatches. A common feature of lesions that are both thermodynamically stabilizing and destabilizing is the alteration of the local DNA flexibility (dynamics). In this review we describe the experimental evidence for altered dynamics from NMR and thermodynamic studies on normal and damaged DNA molecules with respect to recognition by NER and MMR. Based on these data, we propose a model for initial detection of lesions by both NER and MMR that occurs through an indirect readout mechanism of alternative DNA conformations induced by covalent damage and base mismatches.

Animals↗
PubMed2001

Stopped-flow kinetic analysis of replication protein A-binding DNA: damage recognition and affinity for single-stranded DNA reveal differential contributions of k(on) and k(off) rate constants.

Replication protein A (RPA) is a heterotrimeric protein required for many DNA metabolic functions, including replication, recombination, and nucleotide excision repair (NER). We report the pre-steady-state kinetic analysis of RPA-binding DNA substrates using a stopped-flow assay to elucidate the kinetics of DNA damage recognition. The bimolecular association rate, k(on), for RPA binding to duplex DNA substrates is greatest for a 1,3d(GXG), intermediate for a 1,2d(GpG) cisplatin-DNA adduct, and least for an undamaged duplex DNA substrate. RPA displays a decreased k(on) and an increased k(off) for a single-stranded DNA substrate containing a single 1,2d(GpG) cisplatin-DNA adduct compared with an undamaged DNA substrate. The k(on) for RPA-binding single-stranded polypyrimidine sequences appears to be diffusion-limited. There is minimal difference in k(on) for varying length DNA substrates; therefore, the difference in equilibrium binding affinity is mainly attributed to the k(off). The k(on) for a purine-rich 30-base DNA is reduced by a factor of 10 compared with a pyrimidine-rich DNA of identical length. These results provide insight into the mechanism of RPA-DNA binding and are consistent with RPA recognition of DNA-damage playing a critical role in NER.

Base Sequence↗
PubMed1995

Recognition of DNA interstrand cross-links of cis-diamminedichloroplatinum(II) and its trans isomer by DNA-binding proteins.

Recognition and processing by cellular proteins of DNA modified by platinum complexes have been suggested to be relevant to the mechanism of their antitumor activity. Platinum complexes form on DNA various mono- and bifunctional adducts. It has already been described by other authors that intrastrand cross-links formed on DNA by antitumor cis-diamminedichloroplatinum(II) (cisplatin) between neighboring purine residues are recognized by several DNA-binding proteins. In contrast, these proteins do not recognize the intrastrand cross-links formed on DNA by cisplatin or its clinically ineffective trans isomer (transplatin) between nonadjacent base residues. An eventuality heretofore not addressed is that DNA interstrand cross-links (ICLs) of platinum compounds may be recognized by and bound to DNA-binding proteins. DNA probes of 110 base pairs (bp) were constructed containing five equally spaced ICLs of cisplatin or transplatin. These ICLs were formed at specific sites at which these adducts are preferentially formed in natural DNA. Gel electrophoresis mobility shift and competition assays with these probes were used to investigate the specific recognition and binding of the calf thymus HMG1 protein to the DNA ICLs of both platinum isomers. The ICL of antitumor cisplatin was recognized by and bound to the HMG1 protein with a similar affinity as the 1,2-intrastrand d(GpG) cross-link of this drug. The protein binding to the ICL is selective for the DNA modification by cisplatin, but not by chemotherapeutically inactive transplatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents↗
PubMed1994

A framework for the DNA-protein recognition code of the probe helix in transcription factors: the chemical and stereochemical rules.

BACKGROUND: Understanding the general mechanisms of sequence specific DNA recognition by proteins is a major challenge in structural biology. The existence of a 'DNA recognition code' for proteins, by which certain amino acid residues on a protein surface confer specificity for certain DNA bases, has been the subject of much discussion. However, no simple code has yet been established. RESULTS: The principles of DNA recognition can be described at two levels. The 'chemical' rules describe the partnerships between amino acid side chains and DNA bases making favourable interactions in the major groove of DNA. Here I analyze the occurrence of nucleotide-amino acid contacts in previously determined crystal structures of DNA-protein complexes and find that simple rules pertain. I also describe 'stereochemical' rules for the probe helix type of DNA-binding motif found in certain transcription factors including leucine zipper and homeodomain proteins. These are a consequence of the binding geometry, and specify the amino acid and base positions used for the contacts, and the sizes of residues in the contact interface. CONCLUSIONS: The chemical rules can be generalized for any DNA-binding motif, while the stereochemical rules are specific to a particular DNA-binding motif. The recognition code for a particular binding motif can be described by combining the two sets of rules.

Amino Acid Sequence↗
PubMed2001

Recognition and cleavage at the DNA major groove.

DNA recognition agents based on the indole-based aziridinyl eneimine and the cyclopent[b]indole methide species were designed and evaluated. The recognition process involved either selective alkylation or intercalating interactions in the major groove. DNA cleavage resulted from phosphate backbone alkylation (hydrolytic cleavage) and N(7) -alkylation (piperidine cleavage). The formation and fate of the eneimine was studied using enriched 13C NMR spectra and X-ray crystallography. The aziridinyl eneimine specifically alkylates the N(7) position of DNA resulting in direction of the aziridinyl alkylating center to either the 3'- or 5'-phosphate of the alkylated base. The eneimine species forms dimers and trimers that appear to recognize DNA at up to three base pairs. The cyclopent[b]indole quinone methide recognizes the 3'-GT-5' sequence and alkylates the guanine N(7) and the thymine 6-carbonyl oxygen causing the hydrolytic removal of these bases. In summary, new classes of DNA recognition agents are described and the utility of 13C-enrichment and 13C NMR to study DNA alkylation reactions is illustrated.

Alkylating Agents↗
PubMed2006

Structure and substrate recognition of the Escherichia coli DNA adenine methyltransferase.

The structure of the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase in complex with cognate DNA was determined at 1.89 A resolution in the presence of S-adenosyl-L-homocysteine. DNA recognition and the dynamics of base-flipping were studied by site-directed mutagenesis, DNA methylation kinetics and fluorescence stopped-flow experiments. Our data illustrate the mechanism of coupling of DNA recognition and base-flipping. Contacts to the non-target strand in the second (3') half of the GATC site are established by R124 to the fourth base-pair, and by L122 and P134 to the third base-pair. The aromatic ring of Y119 intercalates into the DNA between the second and third base-pairs, which is essential for base-flipping to occur. Compared to previous published structures of bacteriophage T4 Dam, three major new observations are made in E.coli Dam. (1) The first Gua is recognized by K9, removal of which abrogates the first base-pair recognition. (2) The flipped target Ade binds to the surface of EcoDam in the absence of S-adenosyl-L-methionine, which illustrates a possible intermediate in the base-flipping pathway. (3) The orphaned Thy residue displays structural flexibility by adopting an extrahelical or intrahelical position where it is in contact to N120.

DNA Methylation↗
PubMed1983

Preferential recognition of DNA by dimeric RNA polymerase II. An electron microscopic study.

The forms of purified rat liver RNA polymerase II and their functions were analyzed by an electron microscopy. The author found that highly purified RNA polymerase II showed the following distinct two forms; dimeric and monomeric forms. Furthermore, the preferential binding to promotors of lambda dvl template by dimeric enzyme forms was observed. Finally, the role of dimeric RNA polymerase II on DNA template recognition was discussed.

Animals↗
PubMed2000

Recognition of DNA base pair mismatches by a cyclometalated Rh(III) intercalator.

Two cyclometalated complexes of Rh(III), rac-[Rh(ppy)2chrysi]+ and rac-[Rh(ppy)2 phi]+, have been synthesized and characterized with respect to their binding to DNA. The structure of rac-[Rh(ppy)2 phi]Cl.H2O.CH2Cl2 has been determined by X-ray diffraction (monoclinic, P2(1)/c, Z = 4, a = 18.447(3) A, b = 9.770(1) A, c = 17.661(3) A, beta = 94.821(11) degrees, V = 3172.0(8) A3) and reveals that the complex is a distorted octahedron with nearly planar ligands, similar in structure to the DNA mismatch recognition agent [Rh(bpy)2chrysi]3+. The 2-phenylpyridyl nitrogen atoms are shown to be in the axial positions, as a result of trans-directing effects. This tendency simplifies the synthesis and purification of such complexes by limiting the number of possible isomers generated. The abilities of [Rh(ppy)2chrysi]+ and [Rh(ppy)2 phi]+ to bind and, with photoactivation, to cleave DNA have been demonstrated in assays on duplex DNA in the absence and presence of a single CC mismatch. [Rh(ppy)2chrysi]+ was shown upon photoactivation to cleave DNA selectively at the base pair mismatch whereas [Rh(ppy)2 phi]+ cleaves B-DNA nonspecifically. The reactivity of [Rh(ppy)2chrysi]+ was also compared to that of the known mismatch recognition agent [Rh(bpy)2chrysi]3+. Competitive photocleavage studies revealed that a 14-fold excess of [Rh(ppy)2chrysi]+ was required to achieve the same level of binding as that of [Rh(bpy)2chrysi]3+. However, the ratio of damage induced by [Rh(bpy)2chrysi]3+ to that induced by [Rh(ppy)2chrysi]+ is considerably greater than this value, indicating that decreased photoefficiency for the cyclometalated complex must contribute to its significantly attenuated photoreactivity. These cyclometalated intercalators provide the starting points for the design of a new family of metal complexes targeted to DNA.

Base Pair Mismatch↗
PubMed1996

The recognition of DNA damage.

DNA strand breaks are potentially mutagenic and must, therefore, be recognized and repaired. Recent work has identified DNA polymerase epsilon, Ku, and proteins such as DNA-PKcs, Mec1 and Tel1 as key players in DNA damage recognition pathways. Studies on these and other factors have provided important insights into the mechanisms of DNA repair and how DNA damage signals are transduced to the transcription and cell cycle machineries. This work also suggests how deficiencies in DNA damage detection systems can result in genetic instability and cancer.

Animals↗
PubMed1987

On the possible mechanism of recognition of DNA base sequence by steroid hormones.

Geometry of the complex of a steroid hormone, dexamethasone, with a hexanucleotide sequence from the glucocorticoid responsive element d(TGTTCT)2, is optimised here using computer aided geometry simulation with an energy minimization technique. We have also optimised its geometries with genetically modified and arbitrarily chosen DNA sequences. The drug molecule is considered to have both intercalative as well as non-intercalative binding. Comparison of energetics and stereochemical aspects, as well as the H-bonding scheme, is used here to bring out salient features about the mechanism of DNA sequence recognition by steroid hormones.

Base Sequence↗
PubMed1997

The limited strand-separating activity of the UvrAB protein complex and its role in the recognition of DNA damage.

The recognition by Escherichia coli Uvr nucleotide excision repair proteins of a variety of lesions with diverse chemical structures and the presence of helicase activity in the UvrAB complex which can displace short oligonucleotides annealed to single-stranded DNA led to a model in which this activity moves UvrAB along undamaged DNA to damaged sites where the lesion blocks further translocation and the protein-DNA pre-incision complex is formed. To evaluate this mechanism for damage recognition, we constructed substrates with oligonucleotides of different lengths annealed to single-stranded DNA circles and placed a single 2-(acetylamino)fluorene (AAF) lesion either on the oligonucleotide or on the circle. For the substrates with no lesion, the UvrAB complex effectively displaced a 22-mer but not a 27-mer or longer fragments. The presence of AAF on the oligonucleotide significantly increased the release of the 27-mer but oligomers of 30 or longer were not separated. Placing the lesion on the circular strand did not block the release of the fragments. Instead, the releasing activity of UvrAB was stimulated and also depended on the length of the annealed oligonucleotide. These observations do not agree with the predictions of a damage recognition mechanism that depends on helicase-driven translocation. Most likely, the strand-separating activity of UvrAB is a consequence of local changes occurring during the formation of a DNA-protein pre-incision complex at the damaged site and is not due to translocation of the protein along undamaged DNA to locate a lesion.

2-Acetylaminofluorene↗
PubMed2003

MuA transposase separates DNA sequence recognition from catalysis.

Confronted with thousands of potential DNA substrates, a site-specific enzyme must restrict itself to the correct DNA sequence. The MuA transposase protein performs site-specific DNA cleavage and joining reactions, resulting in DNA transposition-a specialized form of genetic recombination. To determine how sequence information is used to restrict transposition to the proper DNA sites, we performed kinetic analyses of transposition with DNA substrates containing either wild-type transposon sequences or sequences carrying mutations in specific DNA recognition modules. As expected, mutations near the DNA cleavage site reduce the rate of cleavage; the observed effect is about 10-fold. In contrast, mutations within the MuA recognition sequences do not directly affect the DNA cleavage or joining steps of transposition. It is well established that the recognition sequences are necessary for assembly of stable, multimeric MuA-DNA complexes, and we find that recognition site mutations severely reduce both the extent and the rate of this assembly process. Yet if the MuA-DNA complexes are preassembled, the first-order rate constants for both DNA cleavage and DNA strand transfer (the joining reaction) are unaffected by the mutations. Furthermore, most of the mutant DNA molecules that are cleaved also complete DNA strand transfer. We conclude that the sequence-specific contacts within the recognition sites contribute energetically to complex assembly, but not directly to catalysis. These results contrast with studies of more orthodox enzymes, such as EcoRI and some other type II restriction enzymes. We propose that the strategy employed by MuA may serve as an example for how recombinases and modular restriction enzymes solve the DNA specificity problem, in that they, too, may separate substrate recognition from catalysis.

Bacteriophage mu↗