Design of a Fluorescent Electrophoretic Mobility Shift Assay Improved for the Quantitative and Multiple Analysis of Protein-DNA Complexes

We describe a protocol for the fluorescent electrophoretic mobility shift assay improved for the quantitative analysis of protein-DNA complexes. Fluorescent-labeled oligonucleotide probes incubated with nuclear proteins were followed by electrophoresis. The signals for protein-DNA complexes were measured and normalized with fluorescent-labeled marker using fragment analysis software. This assay proved reliable measurement and multiple detection of DNA binding proteins.     

A new analytical scale DNA affinity binding assay for analyses of specific protein-DNA interactions.

We describe a rapid analytical assay for identification of proteins binding to specific DNA sequences. The DAPSTER assay (DNA affinity preincubation specificity test of recognition assay) is a DNA affinity chromatography-based microassay that can discriminate between specific and nonspecific protein-DNA interactions. The assay is sensitive and can detect protein-DNA interactions and larger multicomponent complexes that can be missed by other analytical methods. Here we describe in detail the optimization and utilization of the DAPSTER assay to isolate AP-1 complexes and associated proteins in multimeric complexes bound to the AP-1 DNA element.     

Statistical analysis of 15 dimensions in the crystallization space for protein-DNA complexes

Solving the three dimensional structure of a protein-DNA complex is a prerequisite to understand, at the atomic level, the interactions between DNA-binding proteins and their target DNA sequences. Arranging these complexes into an ordered and repetitive network (a crystal, suitable for X-Ray analysis) is a time-limiting empirical step. Although it has been suggested that the crystallization space for protein-DNA complexes is probably smaller than that of non-complexed proteins, a study presenting a detailed and updated analysis of this space is still missing. Here, we analyze the successful crystallization conditions of several hundred protein-DNA complexes and present a bias-free statistical analysis of 15 crystallization parameters that include concentration, temperature, pH, precipitants, salts, divalent cations and polyamines. Our analysis shows that some crystallization parameters are interestingly restricted into narrow intervals. These restrictions could be very helpful in the design of sparse-matrix crystallization screens that target exclusively protein-DNA complexes.     

Binding of the estradiol-receptor complex to reconstituted nucleoacidic protein from calf uterus

Non-histone protein-DNA complexes with acceptor activity for estradiol-receptor complexes were reconstituted from fractionated calf uterine chromatin. Acceptor activity had tissue specificity with target tissue binding exceeding non-target tissue binding. The binding of estradiol-receptor complexes to acceptor sites was dependent on intact non-histone protein-DNA complexes, reconstituted select non-histone proteins, and protein equivalent: DNA reconstitution ratios. [³H]Estradiol-receptor complexes were bound to reconstituted non-histone protein-DNA complexes (i.e., nucleoacidic protein) with a high affinity and with a limited number of binding sites. Fractionation of uterine chromatin non-histone proteins identified two major sets of non-histone proteins which had acceptor activity when reconstituted with DNA. Thus, it seems possible to reconstitute nucleoacidic protein fractions with specific acceptor activity for the calf uterine estrogen receptor.     

Characterization of the molecular and structural properties of the transformed and nuclear aryl hydrocarbon (Ah) receptor complexes by proteolytic digestion

Ligand-dependent differences in the molecular properties of the transformed cytosolic and nuclear aryl hydrocarbon receptor (AhR) were investigated using the proteolytic clipping band shift assay. AhR complexes were incubated with [³²P]dioxin responsive element (DRE) (26-mer) or bromodeoxyuridine (BrdU)-DRE and the resulting protein-DNA or crosslinked protein-DNA complexes were treated with trypsin or V8 protease and analyzed by electrophoresis. The results showed that for several different AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran, 1,2,7,8-tetrachlorodibenzofuran and -naphthoflavone, the pattern of degraded protein-DNA products were similar using transformed cytosolic or nuclear AhR complexes. In contrast, the proteolytic clipping band shift assay showed that there were significant differences in the pattern of degraded protein-DNA products using nuclear AhR complexes derived from mouse Hepa 1c1c7 cells treated with TCDD or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF). The differences detected in this in vitro assay parallel the in vivo and in vitro activities of these compounds in which TCDD is a potent AhR agonist whereas MCDF is a partial AhR agonist and antagonist.     

Geometric analysis and comparison of protein-DNA interfaces: why is there no simple code for recognition?

Structural studies of protein-DNA complexes have shown that there are many distinct families of DNA-binding proteins, and have shown that there is no simple "code" describing side-chain/base interactions. However, systematic analysis and comparison of protein-DNA complexes has been complicated by the diversity of observed contacts, the sheer number of complexes currently available and the absence of any consistent method of comparison that retains detailed structural information about the protein-DNA interface. To address these problems, we have developed geometric methods for characterizing the local structural environment in which particular side-chain/base interactions are observed. In particular, we develop methods for analyzing and comparing spatial relationships at the protein-DNA interface. Our method involves attaching local coordinate systems to the DNA bases and to the C(alpha) atoms of the peptide backbone (these are relatively rigid structural units). We use these tools to consider how the position and orientation of the polypeptide backbone (with respect to the DNA) helps to determine what contacts are possible at any given position in a protein-DNA complex. Here, we focus on base contacts that are made in the major groove, and we use spatial relationships in analyzing: (i) the observed patterns of side-chain/base interactions; (ii) observed helix docking orientations; (iii) family/subfamily relationships among DNA-binding proteins; and (iv) broader questions about evolution, altered specificity mutants and the limits for the design of new DNA-binding proteins. Our analysis, which highlights differences in spatial relationships in different complexes and at different positions in a complex, helps explain why there is no simple, general code for protein-DNA recognition.     

An overview of the structures of protein-DNA complexes

On the basis of a structural analysis of 240 protein-DNA complexes contained in the Protein Data Bank (PDB), we have classified the DNA-binding proteins involved into eight different structural/functional groups, which are further classified into 54 structural families. Here we present this classification and review the functions, structures and binding interactions of these protein-DNA complexes.     

Insights into protein-DNA interactions through structure network analysis

Protein-DNA interactions are crucial for many cellular processes. Now with the increased availability of structures of protein-DNA complexes, gaining deeper insights into the nature of protein-DNA interactions has become possible. Earlier, investigations have characterized the interface properties by considering pairwise interactions. However, the information communicated along the interfaces is rarely a pairwise phenomenon, and we feel that a global picture can be obtained by considering a protein-DNA complex as a network of noncovalently interacting systems. Furthermore, most of the earlier investigations have been carried out from the protein point of view (protein-centric), and the present network approach aims to combine both the protein-centric and the DNA-centric points of view. Part of the study involves the development of methodology to investigate protein-DNA graphs/networks with the development of key parameters. A network representation provides a holistic view of the interacting surface and has been reported here for the first time. The second part of the study involves the analyses of these graphs in terms of clusters of interacting residues and the identification of highly connected residues (hubs) along the protein-DNA interface. A predominance of deoxyribose-amino acid clusters in beta-sheet proteins, distinction of the interface clusters in helix-turn-helix, and the zipper-type proteins would not have been possible by conventional pairwise interaction analysis. Additionally, we propose a potential classification scheme for a set of protein-DNA complexes on the basis of the protein-DNA interface clusters. This provides a general idea of how the proteins interact with the different components of DNA in different complexes. Thus, we believe that the present graph-based method provides a deeper insight into the analysis of the protein-DNA recognition mechanisms by throwing more light on the nature and the specificity of these interactions.     

Capillary electrophoresis-based method to quantitate DNA-protein interactions

A novel, rapid and simple capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) has been developed for the quantitative study of protein-DNA interactions. This method is particularly useful for the study of basic proteins, the most common of the DNA-interacting proteins. To avoid protein stickiness to the capillary walls we have introduced the use of neutral polyacrylamide that requires the use of reverse polarity. Under these conditions, excellent separation of DNA and protein-DNA complexes was obtained without the requirement of a gel matrix, thereby allowing the easy and reliable quantification of protein-DNA affinities. Analysis of the affinities of histones H2B and H4 for a synthetic oligo have been used to demonstrate the reproducibility and accuracy of this method. We have observed that H4 has a higher affinity for DNA than H2B, with half saturation fractions lying in the micromolar range.     

Interaction of wild-type and mutant adeno-associated virus (AAV) Rep proteins on AAV hairpin DNA.

Both the Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) bind to AAV terminal repeat hairpin DNA and can mediate site-specific nicking in vitro at the terminal resolution site (trs) within the terminal repeats. To define the regions of the Rep proteins required for these functions, a series of truncated Rep78 derivatives was created. Wild-type and mutant proteins were synthesized by in vitro translation and analyzed for AAV hairpin DNA binding, trs endonuclease activity, and interaction on hairpin DNA. Amino-terminal deletion mutants which lacked the first 29 or 79 amino acid residues of Rep78 did not bind hairpin DNA, which is consistent with our previous identification of a DNA-binding domain in this region. Progressive truncation of the carboxyl-terminal region of Rep78 did not eliminate hairpin DNA binding until the deletion reached amino acid 443. The electrophoretic mobility of the Rep-specific protein-DNA complexes was inversely related to the molecular weight of the Rep derivative. Analysis of the C-terminal deletion mutants by the trs endonuclease assay identified a region (amino acids 467 to 476) that is essential for nicking but is not necessary for DNA binding. When endonuclease-positive, truncated Rep proteins that bound hairpin DNA were mixed with full-length Rep78 or Rep68 protein in electrophoretic mobility shift assays, a smear of protein-DNA complexes was observed. This smear migrated at an intermediate position with respect to the bands generated by the proteins individually. An antibody recognizing only the full-length protein produced a novel supershift band when included in a mixed binding assay containing Rep68 and a truncated Rep mutant. These experiments suggest that the Rep proteins can form hetero-oligomers on the AAV hairpin DNA.     

Fine structure and activity of discrete RAG-HMG complexes on V(D)J recombination signals

Two lymphoid cell-specific proteins, RAG-1 and RAG-2, initiate V(D)J recombination by introducing DNA breaks at recombination signal sequences (RSSs). Although the RAG proteins themselves bind and cleave DNA substrates containing either a 12-RSS or a 23-RSS, DNA-bending proteins HMG-1 and HMG-2 are known to promote these processes, particularly with 23-RSS substrates. Using in-gel cleavage assays and DNA footprinting techniques, I analyzed the catalytic activity and protein-DNA contacts in discrete 12-RSS and 23-RSS complexes containing the RAG proteins and either HMG-1 or HMG-2. I found that both the cleavage activity and the pattern of protein-DNA contacts in RAG-HMG complexes assembled on 12-RSS substrates closely resembled those obtained from analogous 12-RSS complexes lacking HMG protein. In contrast, 23-RSS complexes containing both RAG proteins and either HMG-1 or HMG-2 exhibited enhanced cleavage activity and displayed an altered distribution of cleavage products compared to 23-RSS complexes containing only RAG-1 and RAG-2. Moreover, HMG-dependent heptamer contacts in 23-RSS complexes were observed. The protein-DNA contacts in RAG-RSS-HMG complexes assembled on 12-RSS or 23-RSS substrates were strikingly similar at comparable positions, suggesting that the RAG proteins mediate HMG-dependent heptamer contacts in 23-RSS complexes. Results of ethylation interference experiments suggest that the HMG protein is positioned 5' of the nonamer in 23-RSS complexes, interacting largely with the side of the duplex opposite the one contacting the RAG proteins. Thus, HMG protein plays the dual role of bringing critical elements of the 23-RSS heptamer into the same phase as the 12-RSS to promote RAG binding and assisting in the catalysis of 23-RSS cleavage.     

Protein-DNA complexes containing DNA-dependent protein kinase in crude extracts from human and rodent cells

The DNA-dependent protein kinase (DNA-PK) is composed of a large catalytic subunit (DNA-PKcs) and a DNA-binding protein, Ku. Cells lacking DNA-PK activity are radiosensitive and are defective in DNA double-strand break repair and V(D)J recombination. Although much information regarding the interactions of Ku with DNA ends is available, relatively little is known about the interaction of DNA-PKcs with DNA-bound Ku. Here we show, using electrophoretic mobility shift assays, that chemical crosslinkers enhance the formation of protein-DNA complexes containing DNA-PKcs, Ku and other proteins in extracts from cells of normal human cell lines. Extracts from cells of the radiosensitive human cell line M059J, which lacks DNA-PKcs, are not competent to form these protein-DNA complexes, while addition of purified DNA-PKcs protein restores complex formation. This assay may be useful for screening for DNA-PK function in cells of human cell lines and for identifying proteins that interact with the DNA-PK-DNA complex. We also show that Ku protein in rodent cells can interact with human DNA-PKcs; however, this assay may be less useful for studying Ku/DNA-PKcs interactions in cells of rodent cell lines due to the low abundance of DNA-PKcs in these cells.     

Specific binding of cruciform DNA structures by a protein from human extracts.

A gel electrophoresis binding assay has been used to probe extracts from cultured human lymphoblasts for proteins that bind cruciform structures in duplex DNA. Proteins have been detected that form complexes with synthetic X- and Y-junctions. Several lines of evidence suggest that binding is specific for DNA structure rather than sequence: (1) X- and Y-structures were bound whereas linear duplexes containing identical DNA sequences were not, (2) Binding occurred with equal efficiency to two X-junctions that were constructed from DNA strands of different sequence, (3) One X-junction successfully competed with another for binding whereas linear duplex DNA did not; and (4) protein-DNA complexes were observed at probe:non-specific competitor DNA ratios of 1:10,000.     

High-specific-activity probes and a high-resolution in-gel photo cross-linking assay for protein-DNA complexes

Photochemical cross-linking has been widely employed to identify proteins interacting with specific sites on DNA. Identification of bound proteins usually relies on transfer of a radiolabel from the DNA to the protein by cross-linking. We set out to fine-map a small viral replication preinitiation complex composed of two protein dimers bound to DNA, the bovine papillomavirus E1E2-ori complex. Here we describe a simple method for generating high-specific-activity probes with a phenyl-azide photoactivatible cross-linking group positioned immediately adjacent to a labeled nucleotide. The method is based on the selective destruction of one 5'-phosphorylated strand of a polymerase chain reaction product with lambda exonuclease and reconstitution of the probe with a phosphorothioate-substituted oligonucleotide, an [alpha-(32)P]dNTP, and thermophilic enzymes. We also developed a high-resolution in-gel cross-linking assay to probe defined protein-DNA complexes. With these methods we have obtained structural information for the papillomavirus E1E2-ori preinitiation complex that would otherwise have been hard to obtain. These approaches should be widely applicable to the study of protein-DNA complexes.     

Intermolecular and intramolecular readout mechanisms in protein-DNA recognition

Protein-DNA recognition plays an essential role in the regulation of gene expression. Regulatory proteins are known to recognize specific DNA sequences directly through atomic contacts (intermolecular readout) and/or indirectly through the conformational properties of the DNA (intramolecular readout). However, little is known about the respective contributions made by these so-called direct and indirect readout mechanisms. We addressed this question by making use of information extracted from a structural database containing many protein-DNA complexes. We quantified the specificity of intermolecular (direct) readout by statistical analysis of base-amino acid interactions within protein-DNA complexes. The specificity of the intramolecular (indirect) readout due to DNA was quantified by statistical analysis of the sequence-dependent DNA conformation. Systematic comparison of these specificities in a large number of protein-DNA complexes revealed that both intermolecular and intramolecular readouts contribute to the specificity of protein-DNA recognition, and that their relative contributions vary depending upon the protein-DNA complexes. We demonstrated that combination of the intermolecular and intramolecular energies derived from the statistical analyses lead to enhanced specificity, and that the combined energy could explain experimental data on binding affinity changes caused by base mutations. These results provided new insight into the relationship between specificity and structure in the process of protein-DNA recognition, which would lead to prediction of specific protein-DNA binding sites.