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.     

Mode of DNA binding by SopA protein of Escherichia coli F plasmid

The binding of SopA to the promoter region of its own gene, in which four copies of SopA's recognition sequence, 5'-CTTTGC-3', are arrayed asymmetrically, was examined in vitro. Titration using electrophoretic mobility shift assay showed that the stoichiometry of SopA protomers to the promoter-region DNA is 4 and that the binding is highly co-operative. The co-operativity was corroborated by EMSA and DNase I footprinting for a number of mutant DNA fragments in which 5'-CTTTGC-3' was changed to 5'-CTTACG-3'. EMSA in the style of circular permutation showed that SopA bends DNA. Mutation at either outermost binding site had a different effect on DNA bending by SopA, reflecting the asymmetry in the arrangement of the binding sites, for which the results of DNase I footprinting were in agreement. Gel filtration chromatography and analytical ultracentrifugation of free SopA showed that the protein can exist as a monomer and oligomers in the absence of ATP. Hence, the results indicate that the co-operativity in SopA's DNA binding is based on its intrinsic protein-protein interaction modified by DNA interaction.     

Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme.

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.     

Roles of IFN consensus sequence binding protein and PU.1 in regulating IL-18 gene expression.

IL-18 is expressed from a variety of cell types. Two promoters located upstream of exon 1 (5'-flanking region) and upstream of exon 2 (intron 1) regulate its expression. Both promoter regions were cloned into pCAT-Basic plasmid to yield p1-2686 for the 5'-flanking promoter and p2-2.3 for the intron 1 promoter. Both promoters showed basal constitutive activity and LPS inducibility when transfected into RAW 264.7 macrophages. To learn the regulatory elements of both promoters, 5'-serial deletion and site-directed mutants were prepared. For the activity of the p1-2686 promoter, the IFN consensus sequence binding protein (ICSBP) binding site between -39 and -22 was critical. EMSA using an oligonucleotide probe encompassing the ICSBP binding site showed that LPS treatment increased the formation of DNA binding complex. In addition, when supershift assays were performed, retardation of the protein-DNA complex was seen after the addition of anti-ICSBP Ab. For the activity of the p2-2.3 promoter, the PU.1 binding site between -31 and -13 was important. EMSA using a PU.1-specific oligonucleotide demonstrated that LPS treatment increased PU.1 binding activity. The addition of PU.1-specific Ab to LPS-treated nuclear extracts resulted in the formation of a supershifted complex. Furthermore, cotransfection of ICSBP or PU.1 expression vector increased p1 promoter activity or IL-18 expression, respectively. Taken together, these results indicate that ICSBP and PU.1 are critical elements for IL-18 gene expression.     

R150A mutant of F TraI relaxase domain: reduced affinity and specificity for single-stranded DNA and altered fluorescence anisotropy of a bound labeled oligonucleotide

F factor TraI is a helicase and a single-stranded DNA nuclease ("relaxase") essential for conjugative DNA transfer. A TraI domain containing relaxase activity, TraI36, was generated previously. Substituting Ala for Arg150 (R150A) of TraI36 reduces in vitro relaxase activity. The mutant has reduced affinity, relative to wild type, for a 3'-TAMRA-labeled 22-base single-stranded oligonucleotide. While both R150A and wild-type TraI36 bind oligonucleotide, only wild type increases steady-state fluorescence anisotropy of the labeled 22-base oligonucleotide upon binding. In contrast, binding by either protein increases steady-state anisotropy of a 3'-TAMRA-labeled 17-base oligonucleotide. Time-resolved intensity data for both oligonucleotides, bound and unbound, require three lifetimes for adequate fits, at least one more than the fluorophore alone. The preexponential amplitude for the longest lifetime increases upon binding. Time-resolved anisotropy data for both oligonucleotides, bound and unbound, require two rotational correlation times for adequate fits. The longer correlation time increases upon protein binding. Correlation times for the protein-bound 17-base oligonucleotide are similar for both proteins, with the longer correlation time in the range of molecular tumbling of the protein-DNA complex. In contrast, protein binding causes less dramatic increases in correlation times for the 22-base oligonucleotide relative to the 17-base oligonucleotide. Binding studies indicate that R150 contributes to recognition of bases immediately 3' to the DNA cleavage site, consistent with the apparent proximity of R150 and the 3' oligonucleotide end. Models in which the R150A substitution alters single-stranded DNA flexibility at the oligonucleotide 3' end or affects fluorophore-DNA or fluorophore-protein interactions are discussed.     

DNA contacts stimulate catalysis by a poxvirus topoisomerase

Eukaryotic type 1B topoisomerases act by forming covalent enzyme-DNA intermediates that transiently nick DNA and thereby release DNA supercoils. Here we present a study of the topoisomerase encoded by the pathogenic poxvirus molluscum contagiosum. Our studies of DNA sites favored for catalysis reveal a larger recognition site than the 5'-(T/C)CCTT-3' sequence previously identified for poxvirus topoisomerases. Separate assays of initial DNA binding and covalent complex formation revealed that different DNA sequences were important for each reaction step. The location of the protein-DNA contacts was mapped by analyzing mutant sites and inosine-substituted DNAs. Some of the bases flanking the 5'-(T/C)CCTT-3' sequence were selectively important for covalent complex formation but not initial DNA binding. Interactions important for catalysis were probed with 5'-bridging phosphorothiolates at the site of strand cleavage, which permitted covalent complex formation but prevented subsequent religation. Kinetic studies revealed that the flanking sequences that promoted recovery of covalent complexes increased initial cleavage instead of inhibiting resealing of the nicked intermediate. These data 1) indicate that previously unidentified DNA contacts can accelerate a step between initial binding and covalent complex formation and 2) help specify models for conformational changes promoting catalysis.     

A nuclear DNA-binding protein expressed during early stages of B cell differentiation interacts with diverse segments within and 3' of the Ig H chain gene cluster.

We have used electrophoretic mobility shift assays (EMSA) to detect B cell lineage-specific nuclear proteins that bind to diverse segments within and 3' of the Ig H chain gene cluster. DNA binding sites include sequences 5' of each of the following C region genes: mu, gamma 1, gamma 2a, epsilon, and alpha. For the most part, these binding sites lie 5' of CH-associated tandem repeats. Binding sites for the same B cell lineage-specific proteins have also been defined in the region 3' of C alpha, close to a recently described B cell-specific enhancer element. Cross-competition of EMSA indicates that the B cell lineage-specific nucleoprotein is indistinguishable from those described previously by others: S alpha-BP and BSAP. Because of the diverse sequences recognized by this protein, we term it NF-HB, B-lineage-specific nuclear factor that binds to Ig H gene segments. EMSA using segments 5' of S gamma 2a (5'S gamma 2a-176) and 3' of C alpha (3' alpha-88) shows multiple binding complexes, two of which are B cell lineage specific. The B cell-specific complex with fastest mobility contains only NF-HB, and the one with slowest mobility contains NF-HB together with a ubiquitous DNA-binding protein(s). The ubiquitous binding protein is different for 5' S gamma 2a-176 and for 3' alpha-88, representing the formation of protein-NF-HB complexes specific for these particular Ig DNA regions. Spleen cells show a single band upon EMSA with either 5'S gamma 2a-176 or 3' alpha-88. Upon LPS stimulation, additional binding complexes of slower mobility were formed resulting in a pattern comparable to those detected in pro-B, pre-B, and B cell lines. We hypothesize that NF-HB may promote physical interactions between the 3' alpha-enhancer and segments of the Ig H gene cluster.