A comparison of the different DNA binding specificities of the bZip proteins C/EBP and GCN4.

The bZip proteins GCN4 and C/EBP differ in their DNA binding specificities: GCN4 binds well to the pseudopalindromic AP1 site 5'-A4T3G2A1C0T1C2'A3'T4'-3' and to the palindromic ATF/CREB sequence 5'-A4T3G2A1-C0*G0'T1'C2'A3'T4'-3'; C/EBP preferentially recognizes the palindromic sequence 5'-A4T3T2G1C0*G0'C1'A2'-A3'T4'-3'. According to the X-ray structures of GCN4-DNA complexes, five residues of the basic region of GCN4 are involved in specific base contacts: asparagine -18, alanine -15, alanine -14, serine -11 and arginine -10 (numbered relative to the start point of the leucine zipper, which we define as +1). In the basic region of C/EBP position -14 is occupied by valine instead of alanine, the other four residues being identical. Here we analyse the role of valine -14 in C/EBP-DNA complex formation. Starting from a C/EBP-GCN4 chimeric bZip peptide which displays C/EBP specificity, we systematically mutated position -14 of its basic region and characterized the DNA binding specificities of the 20 possible different peptides by gel mobility shift assays with various target sites. We present evidence that valine -14 of C/EBP interacts more strongly with thymine 2 than with cytosine 1' of the C/EBP binding site, unlike the corresponding alanine -14 of GCN4, which exclusively contacts thymine 1' of the GCN4 binding sites.     

Identification of C/EBP basic region residues involved in DNA sequence recognition and half-site spacing preference.

C/EBP and GCN4 are basic region-leucine zipper (bZIP) DNA-binding proteins that recognize the dyad-symmetric sequences ATTGCGCAAT and ATGAGTCAT, respectively. The sequence specificities of these and other bZIP proteins are determined by their alpha-helical basic regions, which are related at the primary sequence level. To identify amino acids that are responsible for the different DNA sequence specificities of C/EBP and GCN4, two kinds of hybrid proteins were constructed: GCN4-C/EBP chimeras fused at various positions in the basic region and substitution mutants in which GCN4 basic region amino acids were replaced by the corresponding residues from C/EBP. On the basis of the DNA-binding characteristics of these hybrid proteins, three residues that contribute significantly to the differences in C/EBP and GCN4 binding specificity were defined. These residues are clustered along one face of the basic region alpha helix. Two of these specificity residues were not identified as DNA-contacting amino acids in a recently reported crystal structure of a GCN4-DNA complex, suggesting that the residues used by C/EBP and GCN4 to make base contacts are not identical. A random binding site selection procedure also was used to define the optimal recognition sequences for three of the GCN4-C/EBP fusion proteins. These experiments identify an element spanning the hinge region between the basic region and leucine zipper domains that dictates optimal half-site spacing (either directly abutted for C/EBP or overlapping by one base pair for GCN4) in high-affinity binding sites for these two proteins.     

DNA binding specificity of mutant glucocorticoid receptor DNA-binding domains

Mutation of a small number of amino acids in the DNA-binding domain of the estrogen receptor to the corresponding sequence of the glucocorticoid receptor switches the specificity of the receptor in transactivation assays (Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989) Nature 338, 271-274). We have made the corresponding reciprocal mutations in the context of the glucocorticoid receptor DNA-binding domain and studied the binding of wild type and mutant purified proteins to palindromic glucocorticoid and estrogen response elements as well as to elements of intermediate sequence, using gel mobility shift assays. We show here that a protein with two altered amino acids binds glucocorticoid and estrogen response elements with a low but equal affinity, whereas a protein with an additional changed residue has a high affinity for estrogen response elements but still retains a considerable affinity for glucocorticoid response elements. Using binding sites of intermediate sequence we have further characterized the interaction with DNA. The in vitro DNA binding results are confirmed by in vivo transactivation assays in yeast. Finally we suggest a testable model for amino acid/base pair interactions involved in recognition by the glucocorticoid receptor DNA-binding domain of its target sequence.     

Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis.

The integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) catalyzes site-specific cleavage of 2 bases from the viral long terminal repeat (LTR) sequence yet it binds DNA with little DNA sequence specificity. We have previously demonstrated that the C-terminal half of IN (amino acids 154-288) possesses a DNA binding domain. In order to further characterize this region, a series of clones expressing truncated forms of IN as N-terminal fusion proteins in E.coli were constructed and analyzed by Southwestern blotting. Proteins containing amino acids 1-263, 1-248 and 170-288 retained the ability to bind DNA, whereas a protein containing amino acids 1-180 showed no detectable DNA binding. This defines a DNA binding domain contained within amino acids 180-248. This region contains an arrangement of 9 lysine and arginine residues each separated by 2-4 amino acids (KxxxKxxxKxxxxRxxxRxxRxxxxKxxxKxxxK), spanning amino acids 211-244, which is conserved in all HIV-1 isolates. A clone expressing full-length IN with a C-terminal fusion of 16 amino acids was able to bind DNA comparably to a cloned protein with a free C-terminus, and an IN-specific monoclonal antibody which recognizes an epitope contained within amino acids 264-279 was unable to block DNA binding, supporting the evidence that a region necessary for binding lies upstream of amino acid 264.     

Identification of a short, hydrophilic amino acid sequence critical for origin recognition by the bovine papillomavirus E1 protein

The E1 protein of bovine papillomavirus (BPV) is a site-specific DNA binding protein that recognizes an 18-bp inverted repeat element in the viral origin of replication. Sequence-specific DNA binding function maps to the region from approximately amino acids 140 to 300, and isolated polypeptides containing this region have been shown to retain origin binding in vitro. To investigate the sequence and structural characteristics which contribute to sequence-specific binding, the primary sequence of this region was examined for conserved features. The BPV E1 DNA binding domain (E1DBD) contains three major hydrophilic domains (HR1, amino acids 179-191; HR2, amino acids 218 to 230; and HR3, amino acids 241 to 252), of which only HR1 and HR3 are conserved among papillomavirus E1 proteins. E1DBD proteins with lysine-to-alanine mutations in HR1 and HR3 were severely impaired for DNA binding function in vitro, while a lysine-to-alanine mutation in HR2 had a minimal effect on DNA binding. Mutation of adjacent threonine residues in HR1 (T187 and T188) revealed that these two amino acids made drastically different contributions to DNA binding, with the T187 mutant being severely defective for origin binding whereas the T188 mutant was only mildly affected. Helical wheel projections of HR1 predict that T187 is on the same helical face as the critical lysine residues whereas T188 is on the opposing face, which is consistent with their respective contributions to DNA binding activity. To examine E1 binding in vivo, a yeast one-hybrid system was developed. Both full-length E1 and the E1DBD polypeptide were capable of specifically interacting with the E1 binding site in the context of the yeast genome, and HR1 was also critical for this in vivo interaction. Overall, our results indicate that HR1 is essential for origin binding by E1, and the features and properties of HR1 suggest that it may be part of a recognition sequence that mediates specific E1-nucleotide contacts.     

Analysis of the DNA-binding domain of Escherichia coli DnaA protein

The DNA-binding domain of the Escherichia coli DnaA protein is represented by the 94 C-terminal amino acids (domain 4, aa 374-467). The isolated DNA-binding domain acts as a functional repressor in vivo, as monitored with a mioC:lacZ translational fusion integrated into the chromosome of the indicator strain. In order to identify residues required for specific DNA binding, site-directed and random PCR mutagenesis were performed, using the mioC:lacZ construct for selection. Mutations defective in DNA binding were found all over the DNA-binding domain with some clustering in the basic loop region, within presumptive helix B and in a highly conserved region at the N-terminus of presumptive helix C. Surface plasmon resonance (SPR) analysis revealed different binding classes of mutant proteins. No or severely reduced binding activity was demonstrated for amino acid substitutions at positions R399, R407, Q408, H434, T435, T436 and A440. Altered binding specificity was found for mutations in a 12 residue region close to the N-terminus of helix C. The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temperatures. dnaX suppressors dnaA71 and dnaA721 map to the region close to helix C and bind DNA non-specifically.     

Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1.

To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496.     

Mutational analysis of all conserved basic amino acids in RAG-1 reveals catalytic, step arrest, and joining-deficient mutants in the V(D)J recombinase

Although both RAG-1 and RAG-2 are required for all steps of V(D)J recombination, little is known about the specific contribution of either protein to these steps. RAG-1 contains three acidic active-site amino acids that are thought to coordinate catalytic metal ions. To search for additional catalytic amino acids and to better define the functional anatomy of RAG-1, we mutated all 86 conserved basic amino acids to alanine and evaluated the mutant proteins for DNA binding, nicking, hairpin formation, and joining. We found several amino acids outside of the canonical nonamer-binding domain that are critical for DNA binding, several step arrest mutants with defects in nicking or hairpin formation, and four RAG-1 mutants defective specifically for joining. Analysis of coding joints formed by some of these mutants revealed excessive deletions, frequent use of short sequence homologies, and unusually long palindromic junctional inserts, known as P nucleotides, that result from aberrant hairpin opening. These features characterize junctions found in scid mice, which are deficient for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), suggesting that the RAG proteins and DNA-PKcs perform overlapping functions in coding joint formation. Interestingly, the amino acids that are altered in 12 of our mutants are also mutated in human inherited immunodeficiency syndromes. Our analysis of these mutants provides insights into the molecular mechanisms underlying these disorders.     

DNA-binding domain of bovine papillomavirus type 1 E1 helicase: structural and functional aspects.

The E1 protein of bovine papillomavirus type 1 is a multifunctional enzyme required for papillomaviral DNA replication. It assists in the initiation of replication both as a site-specific DNA-binding protein and as a DNA helicase. Previous work has indicated that at limiting E1 concentrations, the E2 protein is required for efficient E1 binding to the replication origin. In this study, we have defined the domain of the E1 protein required for site-specific DNA binding. Experiments with a series of truncated proteins have shown that the first amino-terminal 299 amino acids contain the DNA-binding domain; however, the coterminal M protein, which is homologous to E1 for the first 129 amino acids, does not bind origin DNA. A series of small internal deletions and substitution mutations in the DNA-binding domain of E1 show that specific basic residues in this region of the protein, which are conserved in all E1 proteins of the papillomavirus family, likely play a direct role in binding DNA and that a flanking conserved hydrophobic subdomain is also important for DNA binding. A region of E1 that interacts with E2 for cooperative DNA binding is also retained in carboxy-terminal truncated proteins, and we show that the ability of full-length E1 to complex with E2 is sensitive to cold. The E1 substitution mutant proteins were expressed from mammalian expression vectors to ascertain whether site-specific DNA binding by E1 is required for transient DNA replication in the cell. These E1 proteins display a range of mutant phenotypes, consistent with the suggestion that site-specific binding by E1 is important. Interestingly, one E1 mutant which is defective for origin binding but can be rescued for such activity by E2 supports significant replication in the cell.     

DNA binding analysis of glucocorticoid receptor specificity mutants.

The glucocorticoid receptor (GR) DNA binding domain consists of several conserved amino acids and folds into two zinc finger-like structures. Previous transactivation experiments indicated that three amino acids residing in this region, Gly, Ser and Val, appear to be critical for target-site discrimination. Based on the solved crystal structure, these residues are at the beginning of an amphipathic alpha-helix that interacts with the DNA's major groove; of these, only valine, however, contacts DNA. In order to examine their functional role directly, we have substituted these residues for the corresponding amino acids from the estrogen receptor (ER), overexpressed and purified the mutant proteins, and assayed their binding specificity and affinity by gel mobility shifts using glucocorticoid or estrogen response elements (GRE or ERE, respectively) as DNA probes. We find that all three residues are indeed required to fully switch GR's specificity to an ERE. The contacting valine in GR is of primary importance. The corresponding residue in ER, alanine, is less important for specificity, while glutamic acid, four amino acids towards the N-terminus, is most critical for ER discrimination. Finally, we show that the GR DNA binding domain carrying all three ER-specific mutations has a significantly higher affinity for an ERE than the ER DNA binding domain itself. We interpret these results in the context of both the data presented here and the crystal structure of the GR DNA binding domain complexed to a GRE.     

Involvement of lysine residues 289 and 291 of the cAMP-responsive element-binding protein in the recognition of the cAMP-responsive element.

The molecular interactions resulting in specific binding of trans-acting factors to distinct cis-acting elements is not well understood. Here we report our attempt to understand the involvement of distinct amino acid residues of the basic domain of cAMP-responsive element-binding protein (delta CREB) in the determination of binding toward the cAMP-responsive element (CRE). Using in vitro mutagenesis, we constructed site-directed mutants of distinct amino acid residues within the DNA contact region of delta CREB. The activities of the mutant proteins were analyzed by gel retardation, methylation interference, and CRE competition analyses. We demonstrate that a single lysine to glutamine substitution at positions 289 and 291 of delta CREB alters the methylation interference pattern of the mutant protein for the CRE site. Additional mutants constructed at these positions demonstrate that only identical basic residues at both positions 289 and 291 of delta CREB can restore the wild type methylation interference pattern of the mutant delta CREB protein for the CRE site. These observations point to the importance of the lysine residues at positions 289 and 291 in the process of CRE binding. In addition, this observation suggests that the symmetrical nature of the CRE site is reflected in the DNA contact region of the protein.