A switch in the mechanism of communication between the two DNA-binding sites in the SfiI restriction endonuclease

While many Type II restriction enzymes are dimers with a single DNA-binding cleft between the subunits, SfiI is a tetramer of identical subunits. Two of its subunits (a dimeric unit) create one DNA-binding cleft, and the other two create a second cleft on the opposite side of the protein. The two clefts bind specific DNA cooperatively to give a complex of SfiI with two recognition sites. This complex is responsible for essentially all of the DNA-cleavage reactions by SfiI: virtually none is due to the complex with one site. The communication between the DNA-binding clefts was examined by disrupting one of the very few polar interactions in the otherwise hydrophobic interface between the dimeric units: a tyrosine hydroxyl was removed by mutation to phenylalanine. The mutant protein remained tetrameric in solution and could bind two DNA sites. But instead of being activated by binding two sites, like wild-type SfiI, it showed maximal activity when bound to a single site and had a lower activity when bound to two sites. This interaction across the dimer interface thus enforces in wild-type SfiI a cooperative transition between inactive and active states in both dimers, but without this interaction as in the mutant protein, a single dimer can undergo the transition to give a stable intermediate with one inactive dimer and one active dimer.     

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.     

Nonidentical DNA-binding sites of endonuclease NaeI recognize different families of sequences flanking the recognition site.

NaeI endonuclease uses a two-site binding mechanism to cleave substrate DNA: reaction-rate studies imply that occupancy of the second DNA site causes an allosteric change in the protein that enables DNA cleavage at the first site [Conrad, M., & Topal, M. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. Measurements of relative binding affinities for 14-base-pair DNA fragments containing the NaeI recognition sequence GCCGGC and various flanking sequences showed that the two DNA-binding sites are not identical. G.C-rich flanking sequences were preferred by the activator binding site, whereas A.T-rich flanking sequences were preferred by the substrate binding site: GGGTGCCGGCAGGG was preferred 8-fold more by the activator site but 14-fold less by the substrate site than TTTCGCCGGCGTTT. Substitution of pyrimidine or 7-deazapurine for purine immediately 3' to GCCGGC reduced DNA affinity for only the activator site by up to 26-fold, implying that the activator DNA-binding site requires N-7 base contacts immediately flanking GCCGGC. The implications of nonidentical DNA-binding sites, one of which binds a specific DNA site to allosterically activate the other, are discussed.     

Chromatin assembly enhances binding to the CYP2B1 phenobarbital-responsive unit (PBRU) of nuclear factor-1, which binds simultaneously with constitutive androstane receptor (CAR)/retinoid X receptor (RXR) and enhances CAR/RXR-mediated activation of the PBRU

Phenobarbital induction of CYP2B genes is mediated by a complex phenobarbital-responsive enhancer (PBRU), which contains a binding site for nuclear factor-1 (NF-1) flanked by two DR-4 nuclear receptor (NR) binding sites for a heterodimer of constitutive androstane receptor (CAR) and retinoid X receptor (RXR). To examine potential interactions between NF-1 and CAR/RXR, binding of purified recombinant proteins to DNA, or to chromatin assembled using Drosophila embryo extract, was examined. NF-1 and CAR/RXR bound simultaneously and independently to the overlapping NF-1 and NR-1 sites; binding of CAR/RXR to the NR-2 site was modestly increased by NF-1 binding; and CAR/RXR bound to a new site in the PBRU region, designated NR-3. Assembly of plasmid DNA into chromatin using Drosophila extract resulted in linearly phased nucleosomes in the PBRU region. The apparent binding affinity of NF-1 was increased by about 10-fold in assembled chromatin compared with DNA, whereas CAR/RXR binding was decreased. As observed for DNA, however, simultaneous, largely independent, binding to the NF-1 and NR sites was observed. CAR-mediated transactivation of the PBRU in cultured cells of hepatic origin was inhibited by mutations in the NF-1 site, and overexpression of NF-1 increased CAR transactivation in HepG2 cells. These studies demonstrate that NF-1 and CAR/RXR can both bind to the PBRU at the same time and that chromatin assembly increases NF-1 binding, which is consistent with previous in vivo footprinting studies in which the NF-1 site was occupied in untreated animals and the NF-1 and flanking NR sites were occupied after phenobarbital treatment. CAR-mediated trans-activation of the PBRU was increased by NF-1, analogous to NF-1 effects on phenobarbital induction in previous transient transfection studies and consistent with mediation of phenobarbital induction by CAR.     

Binding linkage in a telomere DNA-protein complex at the ends of Oxytricha nova chromosomes

Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein-protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (K(D-DNA)=1.4 nM). Another fusion protein, constructed without the C-terminal protein-protein interaction domain of alpha, bound DNA with 200-fold diminished affinity (K(D-DNA)=290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA-protein stability to protein-protein contacts at a remote site may provide a trigger point for DNA-protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase.     

Determinants of high-affinity DNA binding by the glucocorticoid receptor: evaluation of receptor domains outside the DNA-binding domain.

In this study, we have investigated the influence of regions outside the DNA-binding domain of the human glucocorticoid receptor on high-affinity DNA binding. We find that the DNA-binding domain shows a 10-fold lower affinity for a palindromic DNA-binding site than the intact receptor. The N-terminal part of the receptor protein does not influence its DNA-binding affinity, while the C-terminal steroid-binding domain increases the DNA-binding affinity of the receptor molecule. It has previously been shown that both the intact glucocorticoid receptor and the glucocorticoid receptor DNA-binding domain bind to a palindromic glucocorticoid response element on DNA as dimers. It is likely that differences in DNA-binding affinity observed result from protein-protein interactions outside the DNA-binding domain between receptor monomers, as has been shown for the estrogen receptor. We have previously identified a segment involved in protein-protein interactions between DNA-binding domains of glucocorticoid receptors. This, in combination with results presented in this study, suggests that there are at least two sites of contact between receptor monomers bound to DNA. We suggest that the interaction between the DNA-binding domains may act primarily to restrict DNA binding to binding sites with appropriate half-site spacing and that additional stability of the receptor dimer is provided by the interactions between the steroid-binding domains.     

Purified mu EBP-E binds to immunoglobulin enhancers and promoters.

We describe the purification to apparent homogeneity of the murine immunoglobulin heavy-chain (IgH) enhancer-binding protein mu EBP-E from murine plasmacytoma cells by ion exchange and affinity chromatography. Glycerol gradient sedimentation, UV cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirm that mu EBP-E is a 45-kilodalton molecular mass protein. Orthophenanthroline-copper chemical nuclease footprinting with purified protein has identified high-affinity binding sites for mu EBP-E within the IgH enhancer at the previously identified site E and at sites within IgH promoters and in the kappa light-chain enhancer. Equilibrium binding studies indicate that the dissociation constants for mu EBP-E binding to site E within the enhancer and to a binding site within the V1 heavy-chain promoter are quite low, about 2 x 10(-11) M. Comparison of four mu EBP-E recognition sequences detects only limited sequence similarity among binding sites.     

Multiple components are required for sequence recognition of the AP1 site in the gibbon ape leukemia virus enhancer.

At least two subunits contributed to the formation in vitro of a specific complex binding to the AP1 consensus sequence (TGAGTCA) in the gibbon ape leukemia virus (GALV) enhancer in MLA144 cells. This complex can be dissociated on a monomeric GALV oligonucleotide affinity column. One protein, termed the core protein, was retained on the oligonucleotide affinity column. The second protein flowed through the oligonucleotide affinity column and, when alone, did not bind to DNA; however, when present with the core protein, it bound strongly and very specifically to the GALV sequence. MLA144 cells contained only trace amounts of c-fos and c-jun by immunoblot analysis, suggesting that the proteins specifically binding to the GALV AP1 site were distinct from c-fos and c-jun. In addition to the major complex that recognized the GALV element, MLA144 cells contained a minor complex that is chromatographically different from and antigenically related to c-fos. The factor in the flowthrough complemented a human T-cell nuclear extract (Jurkat cell line), which, when alone, had no assayable complex that specifically bound to the GALV enhancer; this complementation gave rise to a specific complex similar to that seen in MLA144 cells. Together, these results suggest that the GALV enhancer can interact with multicomponent protein complexes in a cell-line-specific manner.     

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.     

Mutational analysis of protein binding sites involved in formation of the bacteriophage lambda attL complex.

Bacteriophage lambda site-specific recombination requires the formation of higher-order protein-DNA complexes to accomplish synapsis of the partner attachment (att) sites as well as for the regulation of the integration and excision reactions. The att sites are composed of a core region, the actual site of strand exchange, and flanking arm regions. The attL site consists of two core sites (C and C'), an integration host factor (IHF) binding site (H'), and three contiguous Int binding arm sites (P'1, P'2, and P'3). In this study, we employed bacteriophage P22 challenge phages to determine which protein binding sites participate in attL complex formation in vivo. The C', H', and P'1 sites were critical, because mutations in these sites severely disrupted formation of the attL complex. Mutations in the C and P'2 sites were less severe, and alteration of the P'3 site had no effect on complex formation. These results support a model in which IHF, bound to the H' site, bends the attL DNA so that the Int molecule bound to P'1 also interacts with the C' core site. This bridged complex, along with a second Int molecule bound to P'2, helps to stabilize the interaction of a third Int with the C core site. The results also indicate that nonspecific DNA binding is a significant component of the Int-core interactions and that the cooperativity of Int binding can overcome the effects of mutations in the individual arm sites and core sites.     

Proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer exist in multiple oligomeric forms.

We describe the purification to near homogeneity of proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer. Proteins binding to this site produce four protein-DNA complexes which are distinguished by their mobility in gel retardation assays and their elution properties in an anion exchange column. DNA affinity-purified preparations of three chromatographically separated pools, containing different subsets of the four complexes, each contained three polypeptides of 42.5, 44, and 45 kilodaltons (kDa). UV crosslinking of protein to enhancer DNA demonstrated that site C2-binding activities in the three different pools bound DNA through proteins of similar sizes (about 45 kDa), even though the protein-DNA complexes formed by these binding activities were quite distinct. Gel exclusion chromatography and equilibrium binding analyses indicated that the distinct protein-DNA complexes were due to different oligomeric forms of the individual subunits and that a larger multimeric form bound with high affinity to the heavy-chain enhancer site C2, while a smaller species had a much lower affinity for heavy-chain enhancer sequences. Purified protein has been used to map high-affinity binding sites for site C2-binding proteins within an immunoglobulin heavy-chain promoter and at site KE3 in the kappa light-chain enhancer.     

Iodination of the progesterone receptor from hen oviduct spares the DNA-binding domain

The progesterone receptor from hen oviduct is isolated as a complex of two subunits, A and B. The A protein binds one molecule of progesterone and also binds to DNA with high affinity. The native A protein can be labeled with iodine with no loss of DNA binding activity. Limited Staphylococcus aureus V8 protease digestion of the labeled preparation results in a number of DNA-binding and non-DNA-binding fragments of the receptor. The progesterone-binding domain contains iodine label. However, two low-molecular-weight DNA-binding fragments do not contain iodine label, indicating a lack of susceptible tyrosine residues near the DNA-binding site of the native receptor. The labeled receptor and its fragments will facilitate studies of the isolated DNA-binding and progesterone-binding domains of the hen A protein as well as of the activity of the native receptor in the presence and absence of hormone.