Control of DNA minor groove width and Fis protein binding by the purine 2-amino group

The width of the DNA minor groove varies with sequence and can be a major determinant of DNA shape recognition by proteins. For example, the minor groove within the center of the Fis–DNA complex narrows to about half the mean minor groove width of canonical B-form DNA to fit onto the protein surface. G/C base pairs within this segment, which is not contacted by the Fis protein, reduce binding affinities up to 2000-fold over A/T-rich sequences. We show here through multiple X-ray structures and binding properties of Fis–DNA complexes containing base analogs that the 2-amino group on guanine is the primary molecular determinant controlling minor groove widths. Molecular dynamics simulations of free-DNA targets with canonical and modified bases further demonstrate that sequence-dependent narrowing of minor groove widths is modulated almost entirely by the presence of purine 2-amino groups. We also provide evidence that protein-mediated phosphate neutralization facilitates minor groove compression and is particularly important for binding to non-optimally shaped DNA duplexes.     

HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region.     

Stimulation of p53 DNA binding by c-Abl requires the p53 C terminus and tetramerization

The carboxyl terminus of p53 is a target of a variety of signals for regulation of p53 DNA binding. Growth suppressor c-Abl interacts with p53 in response to DNA damage and overexpression of c-Abl leads to G(1) growth arrest in a p53-dependent manner. Here, we show that c-Abl binds directly to the carboxyl-terminal regulatory domain of p53 and that this interaction requires tetramerization of p53. Importantly, we demonstrate that c-Abl stimulates the DNA-binding activity of wild-type p53 but not of a carboxyl-terminally truncated p53 (p53Delta363C). A deletion mutant of c-Abl that does not bind to p53 is also incapable of activating p53 DNA binding. These data suggest that the binding to the p53 carboxyl terminus is necessary for c-Abl stimulation. To investigate the mechanism for this activation, we have also shown that c-Abl stabilizes the p53-DNA complex. These results led us to hypothesize that the interaction of c-Abl with the C terminus of p53 may stabilize the p53 tetrameric conformation, resulting in a more stable p53-DNA complex. Interestingly, the stimulation of p53 DNA-binding by c-Abl does not require its tyrosine kinase activity, indicating a kinase-independent function for c-Abl. Together, these results suggest a detailed mechanism by which c-Abl activates p53 DNA-binding via the carboxyl-terminal regulatory domain and tetramerization.     

DNA sequence recognition by the antitumor drug ditercalinium

The antitumor drug ditercalinium is a rare example of a noncovalent DNA-binding ligand that forms bisintercalation complexes via the major groove of the double helix. Previous structural studies have revealed that the two connected pyridocarbazolium chromophores intercalate into DNA with the positively charged bis(ethylpiperidinium) linking chain oriented to the wide groove side of the helix. Although the interaction of ditercalinium with short oligonucleotides containing 4-6 contiguous GC base pairs has been examined in detail by biophysical and theoretical approaches, the sequence preference for ditercalinium binding to long DNA fragments that offer a wide variety of binding sites has been investigated only superficially. Here we have investigated both sequence preferences and possible molecular determinants of selectivity in the binding of ditercalinium to DNA, primarily using methods based upon DNase I footprinting. A range of multisite DNA substrates, including several natural restriction fragments and different PCR-generated fragments containing unconventional bases (2,6-diaminopurine, inosine, uridine, 5-fluoro- and 5-methylcytosine, 7-deazaguanine, 7-deazaadenine, and N(7)-cyanoboranoguanine), have been employed to show that ditercalinium selectively recognizes certain GC-rich sequences in DNA and to identify some of the factors which affect its DNA-binding sequence selectivity. Specifically, the footprinting data have revealed that the 2-amino group on the purines or the 5-methyl group on the pyrimidines is not essential for the formation of ditercalinium-DNA complexes whereas the major groove-oriented N(7) of guanine does appear as a key element in the molecular recognition process. The loss of N(7) at guanines but not adenines is sufficient to practically abolish sequence-selective binding of ditercalinium to DNA. Thus, as expected for a major groove binding drug, the N(7) of guanine is normally required for effective complex formation with GC base pairs, but interestingly the substitution of the N(7) with a relatively bulky cyanoborane group does not markedly affect the sequence recognition process. Therefore, the hydrogen bond accepting capability at N(7) of guanines is not sufficient to explain the GC-selective drug-DNA association, and the implications of these findings are considered.     

Binding specificity of the two major DNA-binding proteins in human serum.

The two major DNA-binding proteins of human serum (DNA-binding protein 1 and DNA-binding protein 2) were shown to bind preferentially to single-stranded polynucleotides rich in guanine residues. Equilibrium competition experiments using a nitrocellulose filter assay system containing labeled human lymphocyte DNA and various competing natural and synthetic polynucleotides indicated that both proteins recognized sequences of bases containing a keto group in either position 6 (purines) or 4 (pyrimidines) and that these keto groups must be readily accessible for effective binding to occur. Guanine was shown to be the preferred nucleotide through inhibition experiments using a series of synthetic homopolymers and a series of bacterial DNAs of differing G + C content. The relationship between protein affinity and G + C content was shown to be directly proportional. The equilibrium constants for the binding of the human lymphocyte DNA by both proteins were on the order of 10(-6) M, and the length of the nucleotide sequence necessary for effective binding was found to be 12 to 18 bases using a series of oligomers of poly(dG).     

Uracil interference, a rapid and general method for defining protein-DNA interactions involving the 5-methyl group of thymines: the GCN4-DNA complex.

We describe a novel uracil interference method for examining protein contacts with the 5-methyl group of thymines. The protein of interest is incubated with target DNA containing randomly distributed deoxyuracil substitutions that is generated by carrying out the polymerase chain reaction in the presence of a mixture of TTP and dUTP. After separating DNA-protein complexes away from unbound DNA, the locations of deoxyuracil residues that either do or do not interfere with DNA-binding are determined by cleavage with uracil-N-glycosylase followed by piperidine. Using this uracil interference assay, we show that the methyl groups of the four core thymines, but not the two peripheral thymines, of the optimal binding site (ATG-ACTCAT) are important for high affinity binding of GCN4. Similar, but not identical, results are obtained using KMnO4 interference, another method used for studying protein-DNA interactions involving thymine residues. These observations strongly suggest that GCN4 directly contacts the 5-methyl groups of the four core thymines that lie in the major groove of the target DNA. Besides providing specific structural information about protein-DNA complexes, uracil interference should also be useful for identifying DNA-binding proteins and their target sites in eukaryotic promoter regions.     

Interaction between UV-damaged DNA binding activity proteins and the c-Abl tyrosine kinase

The c-Abl tyrosine kinase is activated by some forms of DNA damage, including ionizing radiation, but not UV radiation. The functions of this activation in the damage response pathways remain obscure. To identify potential targets of c-Abl kinase, we utilized the yeast two-hybrid system to screen a murine cDNA library. One c-Abl binding protein of particular interest was the large subunit (DDB1) of the heterodimeric complex with UV-damaged DNA binding activity (UV-DDB). This complex binds with high specificity to DNA damaged by UV, is absent in a subset of xeroderma pigmentosum group E cells, and is required for global genomic repair of UV-induced damage. The association of c-Abl with DDB1 required the kinase domain of c-Abl and preserved the interaction between DDB1 and the small subunit (DDB2) of the UV-DDB complex. Significantly, overexpression of c-Abl increased tyrosine phosphorylation of DDB2 and suppressed UV-DDB activity. Conversely, a dominant negative, kinase-deficient allele of c-Abl decreased tyrosine phosphorylation of DDB2 and dramatically stimulated UV-DDB activity. These results suggest that one role of c-Abl may be to negatively regulate UV-DDB activity by phosphorylation of DDB2.     

Phosphorylation by cdc2 kinase modulates DNA binding activity of high mobility group I nonhistone chromatin protein

Chromatin high mobility group protein I (HMG-I) is a mammalian nonhistone protein that has been demonstrated both in vitro and in vivo to preferentially bind to A.T-rich sequences of DNA. Recently the DNA-binding domain peptide that specifically mediates the in vitro interaction of high mobility group protein (HMG)-I with the narrow minor groove of A.T-DNA has been experimentally determined. Because of its predicted secondary structure, the binding domain peptide has been called "the A.T hook" motif. Previously we demonstrated that the A.T hook of murine HMG-I protein is specifically phosphorylated by purified mammalian cdc2 kinase in vitro and that the same site(s) are also phosphorylated in vivo in metaphase-arrested cells. We also found that the DNA binding affinity of short synthetic binding domain peptides phosphorylated in vitro by cdc2 kinase was significantly reduced compared with unphosphorylated peptides. Here we extend these findings to intact natural and recombinant HMG-I proteins. We report that the affinity of binding of full-length HMG-I proteins to A.T-rich sequences is highly dependent on ionic conditions and that phosphorylation of intact proteins by cdc2 kinase reduces their affinity of in vitro binding to A.T-DNA by about 20-fold when assayed near normal mammalian physiological salt concentrations. Furthermore, in cell synchronization studies, we demonstrated that murine HMG-I proteins are phosphorylated in vivo in a cell cycle-dependent manner on the same amino acid residues modified by purified cdc2 kinase in vitro. Together these results strongly support the assertion that HMG-I proteins are natural substrates for mammalian cdc2 kinase in vivo and that their cell cycle-dependent phosphorylation by this enzyme(s) significantly modulates their DNA binding affinity, thereby possibly altering their biological function(s).     

Structural requirements for cooperative binding of HMG1 to DNA minicircles

DNA minicircles, where the length of DNA is below the persistence length, are highly effective, preferred, ligands for HMG-box proteins. The proteins bind to them "structure-specifically" with affinities in the nanomolar range, presumably to an exposed widened minor groove. To understand better the basis of this preference, we have studied the binding of HMG1 (which has two tandem HMG boxes linked by a basic extension to a long acidic tail) and Drosophila HMG-D (one HMG box linked by a basic region to a short and less acidic tail), and their HMG-box domains, to 88 bp and 75 bp DNA minicircles. In some cases we see cooperative binding of two molecules to the circles. The requirements for strong cooperativity are two HMG boxes and the basic extension; the latter also appears to stabilize and constrain the complex, preventing binding of further protein molecules. HMG-D, with a single HMG box, does not bind cooperatively. In the case of HMG1, the acidic tail is not required for cooperativity and does not affect binding significantly, in contrast to a much greater effect with linear DNA, or even four-way junctions (another distorted DNA substrate). Such effects could be relevant in the hierarchy of binding of HMG-box proteins to DNA distortions in vivo, where both single-box and two-box proteins might co-exist, with or without basic extensions and acidic tails.     

Binding linkage in a telomere DNA-protein complex at the ends of Oxytricha nova chromosomes

Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein-protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (K(D-DNA)=1.4 nM). Another fusion protein, constructed without the C-terminal protein-protein interaction domain of alpha, bound DNA with 200-fold diminished affinity (K(D-DNA)=290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA-protein stability to protein-protein contacts at a remote site may provide a trigger point for DNA-protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase.     

Transferring the purine 2-amino group from guanines to adenines in DNA changes the sequence-specific binding of antibiotics.

The proposition that the 2-amino group of guanine plays a critical role in determining how antibiotics recognise their binding sites in DNA has been tested by relocating it, using tyrT DNA derivative molecules substituted with inosine plus 2,6-diaminopurine (DAP). Irrespective of their mode of interaction with DNA, such GC-specific antibiotics as actinomycin, echinomycin, mithramycin and chromomycin find new binding sites associated with DAP-containing sequences and are excluded from former canonical sites containing I.C base pairs. The converse is found to be the case for a group of normally AT-selective ligands which bind in the minor groove of the helix, such as netropsin: their preferred sites become shifted to IC-rich clusters. Thus the binding sites of all these antibiotics strictly follow the placement of the purine 2-amino group, which accordingly must serve as both a positive and negative effector. The footprinting profile of the 'threading' intercalator nogalamycin is potentiated in DAP plus inosine-substituted DNA but otherwise remains much the same as seen with natural DNA. The interaction of echinomycin with sites containing the TpDAP step in doubly substituted DNA appears much stronger than its interaction with CpG-containing sites in natural DNA.     

Aph2, a protein with a zf-DHHC motif,interacts with c-Abl and has pro-apoptotic activity

c-Abl is a non-receptor tyrosine kinase implicated in DNA damage-induced cell death and in growth factor receptor signaling. To further understand the function and regulation of c-Abl, a yeast two-hybrid screen was performed to identify c-Abl-interacting proteins. Here we report the identification of Abl-philin 2 (Aph2), encoding a novel protein with a unique cysteine-rich motif (zf-DHHC) and a 53-amino acid stretch sharing homology with the creatine kinase family. The zf-DHHC domain is highly conserved from yeast to human. Two proteins containing this motif, Akr1p and Erf2p, have been characterized in Saccharomyces cerevisiae, both implicated in signaling pathways. Deletion analysis by two-hybrid assays revealed that the N-terminal portion of Aph2 interacts with the C terminus of c-Abl. Aph2 was demonstrated to interact with c-Abl by co-immunoprecipitation assays. Aph2 is expressed in most tissues tested and is localized in the cytoplasm, mainly in the endoplasmic reticulum (ER). The sequences required for ER location reside in the N terminus and the zf-DHHC motif of Aph2. It has been reported that a portion of c-Abl is localized in the ER. We demonstrate here that Aph2 and c-Abl are co-localized in the ER region. Overexpression of Aph2 leads to apoptosis as justified by TUNEL assays, and the induction of apoptosis requires the N terminus. Co-expression of c-Abl and Aph2 had a synergistic effect on apoptosis induction and led to a decreased expression of both proteins, suggesting either that these two proteins are mutually down-regulated or that cells expressing both c-Abl and Aph2 rapidly disappeared from the culture. These results suggest that Aph2 may be involved in ER stress-induced apoptosis in which c-Abl plays an important role.