Base coupling in sequence-specific site recognition by the ETS domain of murine PU.1

The ETS domain of murine PU.1 tolerates a large number of DNA cognates bearing a central consensus 5'-GGAA-3' that is flanked by a diverse combination of bases on both sides. Previous attempts to define the sequence selectivity of this DNA binding domain by combinatorial methods have not successfully predicted observed patterns among in vivo promoter sequences in the genome, and have led to the hypothesis that energetic coupling occurs among the bases in the flanking sequences. To test this hypothesis, we determined, using thermodynamic cycles, the complex stabilities and base coupling energies of the PU.1 ETS domain for a set of 26 cognate variants (based on the lambdaB site of the Ig(lambda)2-4 enhancer, 5'-AATAAAAGGAAGTGAAACCAA-3') in which flanking sequences up to three bases upstream and/or two bases downstream of the core consensus are substituted. We observed that both cooperative and anticooperative coupling occurs commonly among the flanking sequences at all the positions investigated. This phenomenon extends at least three bases in the 5' side and is, at least on our experimental data, due exclusively to pairwise interactions between the flanking bases, and not changes in the local environment of the DNA groove floor. Energetic coupling also occurs between the flanking sides across the core consensus, suggesting long-range conformational effects along the DNA target and/or in the protein. Our data provide an energetic explanation for the pattern of flanking bases observed among in vivo promoter sequences and reconcile the apparent discrepancies raised by the combinatorial experiments. We also discuss the significance of base coupling in light of an indirect readout mechanism in ETS/DNA site recognition.     

Sequence specificity of the core-binding factor.

The core-binding factor (CBF) binds the conserved core motif in mammalian type C retrovirus enhancers. We analyzed the phosphate contacts made by CBF on the Moloney murine leukemia virus enhancer by ethylation interference assay. The phosphate contacts span 9 bp centered around the consensus core site. To examine the sequence preferences for CBF binding, we employed the technique of selected and amplified binding sequence footprinting (T. K. Blackwell and H. Weintraub, Science 250:1104-1110, 1990). The consensus binding site for CBF defined by selected and amplified binding sequence footprinting is PyGPyG GTPy.     

DNA Sequence Determinants Controlling Affinity,Stability and Shape of DNA Complexes Bound by the Nucleoid Protein Fis

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.     

Substrate recognition and induced DNA deformation by transposase at the target-capture stage of Tn10 transposition

The bacterial transposon Tn10 inserts preferentially into sites that conform to a 9 bp consensus sequence: 5' NGCTNAGCN 3'. However, this sequence is not on its own sufficient to confer target specificity as the base-pairs flanking this sequence also contribute significantly to target-site selection. We have performed a series of "contact-probing experiments" to define directly the protein-DNA interactions that govern target-site selection in the Tn10 system. The HisG1 hotspot for Tn10 insertion was the main focus here. We infer that there is a rather broad zone ( approximately 24 bp) of contact between transposase and target DNA in the target-capture complex. This includes base-specific contacts at all of the purine residues in the consensus positions of the target core and primarily backbone contacts out to 7-8 bp in the two flanking regions immediately adjacent to the core. Also, highly localized sites of chemical hypersensitivity are identified that reveal symmetrically disposed deformations in DNA structure in the target-capture complex. Furthermore, the level of strand transfer is shown to be reduced by phosphorothioate substitution of phosphate groups at or close to the sites of target DNA deformation. Interestingly, for one particular target DNA, a mutant form of HisG1 called MutF, the above phosphorothioate inhibition of strand transfer is suppressed by replacing Mg(2+) with Mn(2+). Based on these results a model for sequence-specific target capture is proposed which attempts to define possible relationships between transposase interactions with the target core and flanking sequences, transposase-induced DNA deformation of the target site and divalent metal ion binding to the target-capture complex.     

Requirements for noncovalent binding of vaccinia topoisomerase I to duplex DNA.

Vaccinia DNA topoisomerase binds duplex DNA and forms a covalent adduct at sites containing a conserved sequence element 5'(C/T)CCTT decreases in the scissile strand. Distinctive aspects of noncovalent versus covalent interaction emerge from analysis of the binding properties of Topo(Phe-274), a mutated protein which is unable to cleave DNA, but which binds DNA noncovalently. Whereas DNA cleavage by wild type enzyme is most efficient with 'suicide' substrates containing fewer than 10 base pairs distal to the scissile bond, optimal noncovalent binding by Topo(Phe-274) requires at least 10-bp of DNA 3' of the cleavage site. Thus, the region of DNA flanking the pentamer motif serves to stabilize the noncovalent topoisomerase-DNA complex. This result is consistent with the downstream dimensions of the DNA binding site deduced from nuclease footprinting. Topo(Phe-274) binds to duplex DNA lacking the consensus pentamer with 7-10-fold lower affinity than to CCCTT-containing DNA.     

DNA sequence preferences of several AT-selective minor groove binding ligands.

We have examined the interaction of distamycin, netropsin, Hoechst 33258 and berenil, which are AT-selective minor groove-binding ligands, with synthetic DNA fragments containing different arrangements of AT base pairs by DNase I footprinting. For fragments which contain multiple blocks of (A/T)4 quantitative DNase I footprinting reveals that AATT and AAAA are much better binding sites than TTAA and TATA. Hoechst 33258 shows that greatest discrimination between these sites with a 50-fold difference in affinity between AATT and TATA. Alone amongst these ligands, Hoechst 33258 binds to AATT better than AAAA. These differences in binding to the various AT-tracts are interpreted in terms of variations in DNA minor groove width and suggest that TpA steps within an AT-tract decrease the affinity of these ligands. The behaviour of each site also depends on the flanking sequences; adjacent pyrimidine-purine steps cause a decrease in affinity. The precise ranking order for the various binding sites is not the same for each ligand.     

Specific and nonspecific interactions of integration host factor with oligo DNAs as revealed by circular dichroism spectroscopy and filter binding assay.

Binding specificity of integration host factor (IHF) to oligo DNAs has been studied by circular dichroism (CD) spectroscopy and filter binding experiment. CD difference spectra of IHF-DNA complexes demonstrated that a conformational change in DNA was induced by binding of IHF when DNA had a consensus sequence for the binding sites of IHF, but that such conformational change was not observed for consensus DNA 20 mer as well as nonconsensus DNA 45 mer. Dissociation constants for IHF-DNA complexes determined by filter binding assay showed that IHF has indeed stronger affinity to DNA with the consensus binding site than to nonconsensus DNA, but the difference in its affinity between consensus and nonconsensus DNAs was rather small, 3.4-fold. It was, therefore, concluded that the flanking regions of the consensus sequence are important for the specific binding of IHF and that its binding specificity is well characterized by the induced conformational change in DNA rather than by dissociation constants for IHF-DNA complexes.