Interaction of superhelical DNA with the nonhistone protein HMG1

The interaction between nonhistone chromosomal protein HMG1 and plasmid DNA was studied by optical and hydrodynamical methods. The recombinant protein HMG1 produced by yeast Pichia pastoris strain was used. We have shown that according to the CD spectra local conformational changes in DNA helix occur in the region of DNA-protein interaction. These changes are most significant at r < 3 (w/w). Both gel-shift assay and ultracentrifugation, as well as CD data, indicate that protein-protein interactions between HMG1 molecules play a major role in the formation of DNA-protein complexes. It is suggested that the protein C-terminus may affect HMG1-DNA binding not only by a direct interaction with DNA helix, but also by protein-protein interactions.     

A blotting method for monitoring the formation of chemically induced DNA-protein complexes

The formation and identification of DNA-protein crosslinks are usually detected by filter binding assays such as alkaline elution. We describe a modified blotting method to selectively identify DNA-protein complexes (DPCs) formed in vitro by either Cr3+ ion or formaldehyde. This protocol allows DPC formation in vitro to be assayed with various chemical agents, requires minimal usage of radioactivity, and is performed in a shorter time frame than that commonly used to resolve DPCs from free proteins and unbound DNA.     

Trax is a component of the Translin-containing RNA binding complex

Translin is a nucleic acid binding protein that has been implicated in regulating the targeting and translation of dendritic RNA. In previous studies, we found that Translin and its partner protein, Trax, are components of a gel-shift complex that is highly enriched in brain extracts. In those studies, we employed a DNA oligonucleotide, GS1, as a probe to label the complex. Translin has also been identified as a component of a gel-shift complex detected using an RNA oligonucleotide probe, derived from the 3' UTR of protamine-2 mRNA. Although we had assumed that these probes labeled the same complex, recent studies indicate that association of Trax with Translin suppresses its RNA binding activity. As these findings challenge this assumption and suggest that the native RNA binding complex does not contain Trax, we have re-examined this issue. We have found that the gel-shift complexes labeled with either GS1 or protamine-2 probes are "supershifted" by addition of Trax antibodies, indicating that both are heteromeric Translin/Trax complexes. In addition, cross-competition studies provide additional evidence that these probes label the same complex. Furthermore, analysis of recombinant Translin/Trax complexes generated by co-transfection of Trax with Translin in hEK293T demonstrates that they are labeled with either probe. Although recombinant Translin forms a homomeric nucleic acid binding complex in vitro, our findings indicate that both Trax and Translin are components of the native gel-shift complex labeled with either GS1 or protamine-2 probes.     

Rice proteins that bind single-stranded G-rich telomere DNA

In this work, we have identified and characterized proteins in rice nuclear extracts that specifically bind the single-stranded G-rich telomere sequence. Three types of specific DNA-protein complexes (I, II, and III) were identified by gel retardation assays using synthetic telomere substrates consisting of two or more single-stranded TTTAGGG repeats and rice nuclear extracts. Since each complex has a unique biochemical property and differs in electrophoretic mobility, at least three different proteins interact with the G-rich telomere sequences. These proteins are called rice G-rich telomere binding protein (RGBP) and none of them show binding affinity to double-stranded telomere repeats or single-stranded C-rich sequence. Changing one or two G's to C's in the TTTAGGG repeats abolishes binding activity. RGBPs have a greatly reduced affinity for human and Tetrahymena telomeric sequence and do not efficiently bind the cognate G-rich telomere RNA sequence UUUAGGG. Like other telomere binding proteins, RGBPs are resistant to high salt concentrations. RNase sensitivity of the DNA-protein interactions was tested to investigate whether an RNA component mediates the telomeric DNA-protein interaction. In this assay, we observed a novel complex (complex III) in gel retardation assays which did not alter the mobilities or the band intensities of the two pre-existing complexes (I and II). The complex III, in addition to binding to telomeric sequences, has a binding affinity to rice nuclear RNA, whereas two other complexes have a binding affinity to only single-stranded G-rich telomere DNA. Taken together, these studies suggest that RGBPs are new types of telomere-binding proteins that bind in vitro to single-stranded G-rich telomere DNA in the angiosperms.     

Identification of tyrosine 204 as the photo-cross-linking site in the DNA-EcoRI DNA methyltransferase complex by electrospray ionization mass spectrometry

We describe a highly sensitive strategy combining laser-induced photo-cross-linking and HPLC-based electrospray ionization mass spectrometry to identify amino acid residues involved in protein-DNA recognition. The photoactivatible cross-linking thymine isostere, 5-iodoracil, was incorporated at a single site within the sequence recognized by EcoRI DNA methyltransferase (GAATTC). UV irradiation of the DNA-protein complex at 313 nm results in a >60% cross-linking yield. SDS-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the covalent cross-linked complex. The total mass is consistent with covalent bond formation between one strand of DNA and the protein with 1:1 stoichiometry. Protease digestion of the cross-linked complex yields several peptide-DNA adducts that were purified by anion-exchange column chromatography. A combination of mass spectrometric analysis and amino acid sequencing revealed that tyrosine 204 was cross-linked to the DNA. Electrospray mass spectrometric analysis of the peptide-nucleoside adduct confirmed this assignment. Tyrosine 204 resides in a peptide motif previously thought to be involved in AdoMet binding and methyl transfer. Thus, amino acids within loop segments but outside of "DNA binding" motifs can be critical to DNA recognition. Our method provides an accurate characterization of picomole quantities of DNA-protein complexes.     

Identification of the protein components of mismatch binding complexes in human cells using a gel-shift assay.

In eukaryotes, mismatch recognition is thought to be mediated by two heterodimers, hMutSalpha (hMSH2+hMSH6), which preferentially binds to base-base mismatches and hMutSbeta (hMSH2+hMSH3), which binds to insertion/deletion loops. We studied these mismatch binding activities in several human cell lines with a gel-shift assay using various mismatch oligonucleotides as substrates. Both hMutSalpha and hMutSbeta activities could be detected in various human cell lines. In cells with amplified copies of the hMSH3 gene, a large increase in hMutSbeta and a reduction in hMutSalpha were observed. To identify the composition of each mismatch binding complex, the protein-DNA complexes were transferred from gel-shift polyacrylamide gel to a polyvinylidene difluoride membrane and were subjected to immunoblot analysis with an enhanced chemiluminescence protein detection system. The results clearly demonstrated that hMutSalpha detected by the gel-shift assay was composed of hMSH2 and hMSH6, while hMutSbeta was composed of hMSH2 and hMSH3. Our data, therefore, support a model whereby formation of hMutSalpha and hMutSbeta is mutually regulated. Combination of a gel-shift assay with immunoblotting (shift-Western assay) proved to be a highly sensitive technique and should be useful for studying the interactions between DNA and binding proteins, including DNA mismatch recognition.     

Thermodynamic dissection of the polymerizing and editing modes of a DNA polymerase

DNA polymerases with intrinsic proofreading activity interact with DNA primer/templates in two distinct modes, corresponding to the complexes formed during the 5'-3' polymerization or 3'-5' editing of a nascent DNA chain. Thermodynamic measurements designed to quantify the energetic contributions of individual DNA-protein contacts in either the polymerizing or editing complexes are complicated by the fact that both species exist in solution and are not resolved in conventional DNA-protein binding assays. To overcome this problem, we have developed a new binding analysis that combines information from steady-state and time-resolved fluorescence experiments and uses the Klenow fragment of Escherichia coli DNA polymerase I (KF) and fluorescently labeled primer/template oligonucleotides as a model polymerase-DNA system. Steady-state fluorescence titrations are used to evaluate the overall affinity of KF for the primer/template, while time-resolved fluorescence anisotropy is used to quantify the equilibrium fractions of the primer/template bound in the polymerizing and editing modes. From a combined analysis of both data, the equilibrium constant and hence standard free energy change associated with each binding mode can be obtained unequivocally. This method is initially used to determine the equilibrium constants describing binding of a correctly base-paired primer/template to the 5'-3' polymerase and 3'-5' exonuclease sites of KF. It is then extended to quantify the extent to which these parameters are affected by the introduction of mismatches into the primer/template, and by rearrangement of specific side-chains in the exonuclease domain of the protein. While these perturbants were originally designed to demonstrate the utility of our new approach, they are also relevant in their own right since they have helped identify some hitherto unknown determinants of polymerase fidelity.     

Substitution of basic amino acids in the basic region stabilizes DNA binding by E12 homodimers.

The E2A gene encodes two alternatively spliced products, E12 and E47. The two proteins differ in their basic helix-loop-helix motifs (bHLH), responsible for DNA binding and dimerization. Although both E12 and E47 can bind to DNA as heterodimers with tissue-specific bHLH proteins, E12 binds to DNA poorly as homodimers. An inhibitory domain in E12 has previously been found to prevent E12 homodimers from binding to DNA. By measuring the dissociation rates using filter binding and electrophoretic mobility shift assays, we have shown here that the inhibitory domain interferes with DNA binding by destabilizing the DNA-protein complexes. Furthermore, we have demonstrated that substitution of basic amino acids (not other amino acids) in the DNA-binding domain of E12 can increase the intrinsic DNA-binding activity of E12 and stabilize the binding complexes, thus alleviating the repression from the inhibitory domain. This ability of basic amino acids to stabilize DNA-binding complexes may be of biological significance in the case of myogenic bHLH proteins, which all possess two more basic amino acids in their DNA binding domain than E12. To function as heterodimers with E12, the myogenic bHLH proteins may need stronger DNA binding domains.     

Structure of DNA complexes with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions: 1. Circular dichroism spectroscopy

Mechanisms of interaction of DNA with nonhistone chromosomal protein HMGB1 and linker histone H1 have been studied by means of circular dichroism and absorption spectroscopy. Both proteins are located in the internucleosomal regions of chromatin. It is demonstrated that the properties of DNA-protein complexes depend on the protein content and cannot be considered as a mere summing up of the effects of individual protein components. Interaction of the HMGB1 and H1 proteins is shown with DNA to be cooperative rather than competitive. Lysine-rich histone H1 facilitates the binding of HMGB1 to DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino acid residues in the C-terminal domain of HMGB1. The observed joint action of HMGB1 and H1 stimulates DNA condensation with the formation of anisotropic DNA-protein complexes with typical ψ-type CD spectra. Structural organization of the complexes depends not only on DNA-protein interactions but also on interaction between the HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the mode of interactions between components in the triple DNA-HMGB1-H1 complex. The binding of Mn²⁺ ions weakens DNA-protein interactions and strengthens protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.     

A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.

The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified. This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promotor, but not with a CAP-insensitive mutant. Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.     

Mutational analysis of archaeal histone-DNA interactions

Site-specific mutagenesis of the hmfB gene cloned from the archaeon Methanothermus fervidus, followed by expression in Escherichia coli, has been used to generate approximately 90 recombinant (r) variants of the archaeal histone HMfB. The abilities of these variants to form stable archaeal nucleosome-containing complexes with linear pBR322 DNA, and with an 89 bp restriction fragment of this DNA have been determined. Variants that failed to form such complexes, based on negative gel-shift assays, had substitutions at the N terminus or within the alpha1, L1 and L2 regions of the rHMfB histone fold, at sites predicted to be homologous to eucaryal histone fold residues that contact the DNA in the eucaryal nucleosome. Variants that failed to give gel shifts were further assayed for their abilities to facilitate ligase-catalyzed circularization of a linear 88 bp DNA molecule, and to reduce the ellipticity of a DNA solution at 275 nm (theta(275)). Consistent with cooperative but independent sites of DNA binding, a combination of three residue substitutions, one each in alpha1, L1 and L2, was required to generate a rHMfB variant with no detectable DNA binding based on gel shift, circularization and theta(275) reduction assays.     

Analysis of DNA-protein complexes induced by chemical carcinogens.

DNA-protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis-platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA-protein complex. An antiserum which was raised to chromate-induced DNA-protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA-protein complexes formed by cis-platinum, UV, formaldehyde, and chromate. Other work in this area, involving studying whether DNA-protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA-protein complexes, the stability and repair of these lesions, and characterization of DNA-protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA-protein complexes and these findings are also reviewed.     

The two core sequences of the adenovirus E1A inducible E4 promoter are required for the formation of a specific DNA-protein complex

We have previously demonstrated that the Adenovirus 2 (Ad2) E4 promoter is activated by an E1A gene product through an inducible enhancer. We now show that several DNA-protein complexes can be identified by gel-shift assay; the formation of one of these complexes involves the two core sequences previously found critical to the promoter activity.     

Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding

PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in biophysical analysis of DNA and DNA-protein systems due to limited knowledge pertaining to its physical properties and characteristics of DNA binding. Here we have investigated PicoGreen binding to DNA to reveal the origin and mode of PicoGreen/DNA interactions, in particular the role of electrostatic and nonelectrostatic interactions in formation of the complex, as well as demonstrating minor groove binding specificity. Analysis of the fluorescence properties of free PicoGreen, the diffusion properties of PG/DNA complexes, and the excited-state lifetime changes upon DNA binding and change in solvent polarity, as well as the viscosity, reveal that quenching of PicoGreen in the free state results from its intramolecular dynamic fluctuations. On binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule, resulting in a >1000-fold enhancement in its fluorescence. Based on the results of this study, a model of PicoGreen/DNA complex formation is proposed.