Real-time DNA binding measurements of the ETS1 recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms.

The sequence-specific DNA binding of recombinant p42 and p51 ETS1 oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activity. The kinetics of sequence-specific DNA binding was measured using real-time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real-time binding of p42 and p51 ETS1 displayed significant differences in kinetic behavior. p51 ETS1 is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETS1 exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETS1 DNA binding states are characterized by rapid turnover, whereas the p42 ETS1 DNA binding states are 4-20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETS1 with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETS1 as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETS1 by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETS1 sequence-specific DNA binding occurs through phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.     

Isolation and sequence-specific DNA binding of the Antennapedia homeodomain.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was overproduced in a T7 expression vector in Escherichia coli. The corresponding polypeptide of 68 amino acids was purified to homogeneity. The homeodomain was analysed by ultracentrifugation and assayed for DNA binding. The secondary structure of the isolated homeodomain was determined by nuclear magnetic resonance spectroscopy. DNA-binding studies indicate that the isolated homeodomain binds to DNA in vitro. It selectively binds to the same sites as a longer Antp polypeptide and a full-length fushi tarazu (ftz) protein. Therefore, the homeodomain represents the DNA-binding domain of the homeotic proteins.     

Efficient pH-independent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNA.

The synthesis and DNA binding properties of bis-PNA (peptide nucleic acid) are reported. Two PNA segments each of seven nucleobases in length were connected in a continuous synthesis via a flexible linker composed of three 8-amino-3,6-dioxaoctanoic acid units. The sequence of the first strand was TCTCTTT (C- to N-terminal), while the second strand was TTTCTCT or TTTJTJT, where J is pseudoisocytosine. These bis-PNAs form triple-stranded complexes of somewhat higher thermal stability than monomeric PNA with complementary oligonucleotides and the thermal melting transition shows very little hysteresis. When the J base is placed in the strand parallel to the DNA complement ('Hoogsteen strand'), the DNA binding was pH independent. The bis-PNAs were also superior to monomeric PNAs for targeting double-stranded DNA by strand invasion.     

A geminivirus replication protein is a sequence-specific DNA binding protein.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. The A component encodes the only viral protein, AL1, that is required for viral replication. We showed that AL1 interacts specifically with TGMV A and B DNA by using an immunoprecipitation assay for AL1:DNA complex formation. In this assay, a monoclonal antibody against AL1 precipitated AL1:TGMV DNA complexes, whereas an unrelated antibody failed to precipitate the complexes. Competition assays with homologous and heterologous DNAs established the specificity of AL1:DNA binding. AL1 produced by transgenic tobacco plants and by baculovirus-infected insect cells exhibited similar DNA binding activity. The AL1 binding site maps to 52 bp on the left side of the common region, a 235-bp region that is highly conserved between the two TGMV genome components. The AL1:DNA binding site does not include the putative hairpin structure that is conserved in the common regions or the equivalent 5' intergenic regions of all geminiviruses. These studies demonstrate that a geminivirus replication protein is a sequence-specific DNA binding protein, and the studies have important implications for the role of this protein in virus replication.     

Zfy1 encodes a nuclear sequence-specific DNA binding protein

Zfy1 is a mouse Y chromosomal gene encoding a zincfinger protein which is thought to have some function during spermatogenesis. Here we show that, when introduced into tissue culture cells, Zfy1 is targeted to the nucleus. Two independent signals are present within the protein for nuclear localization. This nuclear Zfy1 protein is able to bind strongly to DNA-cellulose and, using site-selection assays, we have identified specific Zfy1 DNA binding sites. Taken together these results suggest that Zfy1 is a nuclear-located sequence-specific DNA binding protein which functions during spermatogenesis.     

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.     

Two domains of the epstein-barr virus origin DNA-binding protein, EBNA1, orchestrate sequence-specific DNA binding

The EBNA1 (for Epstein-Barr nuclear antigen 1) protein of Epstein-Barr virus governs the replication and partitioning of the viral genomes during latent infection by binding to specific recognition sites in the viral origin of DNA replication. The crystal structure of the DNA binding portion of the EBNA1 protein revealed that this region comprises two structural motifs; a core domain, which mediates protein dimerization and is structurally homologous to the DNA binding domain of the papillomavirus E2 protein, and a flanking domain, which mediated all the observed sequence-specific contacts. To test the possibility that the EBNA1 core domain plays a role in sequence-specific DNA binding not revealed in the crystal structure, we examined the effects of point mutations in potential hydrogen bond donors located in an alpha-helix of the EBNA1 core domain whose structural homologue in E2 mediates sequence-specific DNA binding. We show that these mutations severely reduce the affinity of EBNA1 for its recognition site, and that the core domain, when expressed in the absence of the flanking domain, has sequence-specific DNA binding activity. Flanking domain residues were also found to contribute to the DNA binding activity of EBNA1. Thus, both the core and flanking domains of EBNA1 play direct roles in DNA recognition.     

Porphyrin-linked oligonucleotides. Synthesis and sequence-specific modification of ssDNA

Oligonucleotide derivatives bearing hemin and deuterohemin groups were synthesized. The derivatives efficiently react with the complementary nucleotide sequence in ssDNA forming covalent adducts and piperidine-labile sites. In the case of the deuterohemin derivative, some direct cleavage of the target DNA occurs.     

Redox state regulates binding of p53 to sequence-specific DNA, but not to non-specific or mismatched DNA.

Redox modulation of wild-type p53 plays a role in sequence-specific DNA binding in vitro . Reduction produces a DNA-binding form of the protein while oxidation produces a non-DNA-binding form. Primer extension analysis reveals that increasing concentrations of reduced p53 result in enhanced protection of the consensus sequence, while increasing concentrations of oxidized p53 confer minimal protection of the consensus sequence. DNA binding by oxidized p53 is, therefore, not sequence-specific. In contrast, there is no observable difference in the binding of oxidized p53 and reduced p53 to double-stranded non-specific or mismatched DNA in gel mobility shift assays. Both forms of p53 bind equally well, suggesting that redox modulation of p53 does not play a role in its binding to non-specific or mismatched DNA. In view of the in vitro evidence that redox state influences the sequence-specific DNA-binding of p53, we have examined the effect of oxidative stress on the in vivo ability of p53 to bind to and transactivate PG13-CAT, a reporter construct containing multiple copies of the p53 consensus binding site linked to the chloramphenicol acetyltransferase gene. Hydrogen peroxide treatment of cells cotransfected with p53 results in a marked decrease in CAT activity, suggesting that oxidation of p53 decreases the ability of the protein to bind to consensus DNA and transactivate target genes in vivo.