Libraries for genomic SELEX

An increasing number of proteins are being identified that regulate gene expression by binding specific nucleic acidsin vivo. A method termed genomic SELEX facilitates the rapid identification of networks of protein-nucleic acid interactions by identifying within the genomic sequences of an organism the highest affinity sites for any protein of the organism. As with its progenitor, SELEX of random-sequence nucleic acids, genomic SELEX involves iterative binding, partitioning, and amplification of nucleic acids. The two methods differ in that the variable region of the nucleic acid library for genomic SELEX is derived from the genome of an organism. We have used a quick and simple method to construct Escherichia coli, Saccharomyces cerevisiae, and human genomic DNA PCR libraries that can be transcribed with T7 RNA polymerase. We present evidence that the libraries contain overlapping inserts starting at most of the positions within the genome, making these libraries suitable for genomic SELEX.     

Cellular protein interactions with herpes simplex virus type 1 oriS

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.     

Structure of the Escherichia coli primase/single-strand DNA-binding protein/phage G4oric complex required for primer RNA synthesis

Escherichia coli primase/SSB/single-stranded phage G4oric is a simple system to study how primase interacts with DNA template to synthesize primer RNA for initiation of DNA replication. By a strategy of deletion analysis and antisense oligonucleotide protection on small single-stranded G4oric fragments, we have identified the DNA sequences required for binding primase and the critical location of single-strand DNA-binding (SSB) protein. Together with the previous data, we have defined the structure of the primase/SSB/G4oric priming complex. Two SSB tetramers bind to the G4oric secondary structure, which dictates the spacing of 3' and 5' bound adjacent SSB tetramers and leaves SSB-free regions on both sides of the stem-loop structure. Two primase molecules then bind separately to specific DNA sequences in the 3' and 5' SSB-free G4oric regions. Binding of the 3' SSB tetramer, upstream of the primer RNA initiation site, is also necessary for priming. The generation of a primase-recognition target by SSB phasing at DNA hairpin structures may be applicable to the binding of initiator proteins in other single-stranded DNA priming systems. Novel techniques used in this study include antisense oligonucleotide protection and RNA synthesis on an SSB-melted, double-stranded DNA template.     

Cooperative non-specific DNA binding by octamerizing lambda cI repressors: a site-specific thermodynamic analysis

Relationships between dimerization and site-specific binding have been characterized previously for wild-type and mutant cI repressors at the right operator (OR) of bacteriophage lambda DNA. However, the roles of higher-order oligomers (tetramers and octamers) that are also formed from these cI molecules have remained elusive. In this study, a clear correlation has been established between repressor oligomerization and non-specific DNA-binding activity. A modification of the quantitative DNase I footprint titration technique has been used to evaluate the degree of saturation of non-specific, OR-flanking lambda DNA by cI repressor oligomers. With the exception of one mutant, only those repressors capable of octamerizing were found to exhibit non-specific DNA-binding activity. The non-specific interaction was accurately modeled using either a one-dimensional, univalent, site-specific Ising lattice approximation, or a more traditional, multivalent lattice approach. It was found that non-specific DNA-binding by repressor oligomers is highly cooperative and energetically independent from site-specific binding at OR. Furthermore, the coupling free energy resolved for non-specific binding was similar to that of site-specific binding for each repressor, suggesting that similar structural elements may mediate the cooperative component of both binding processes. It is proposed that the state of assembly of the repressor molecule modulates its relative affinity for specific and non-specific DNA sequences. These specificities are allosterically regulated by the transmission of assembly-state information from the C-terminal domain, which mediates self-association and cooperativity, to the N-terminal domain, which primarily mediates DNA-binding. While dimers have a high affinity for their cognate sites within OR, tetramers and octamers may preferentially recognize non-specific DNA sequences. The concepts and findings developed in this study may facilitate quantitative characterization of the relationships between specific, and non-specific binding in other systems that utilize multiple modes of DNA-binding cooperativity.     

Selective recognition of tandemly repeated DNA sequences by homo- and heterodimers of short peptides

The non-covalent and specific protein-protein interaction is critical to the specificity and the cooperativity of the DNA-binding by protein dimers. We have designed and synthesized three sets of peptide dimers with covalent- or noncovalent artificial dimerization modules to elucidate the structural and thermodynamic aspects for the sequence-specific DNA-binding by protein dimers. We have investigated the DNA binding of covalent dimer, noncovalent homodimer and noncovalent heterodimer with specific or nonspecific DNA sequences by gel mobility shift assay. Although the amino acid sequence of DNA-binding region of these peptide dimers are the same, the selectivity and the cooperativity of DNA binding by these peptide dimers were found to be different.     

Construction and characterization of a quadruplex DNA selective single-chain autoantibody from a viable motheaten mouse hybridoma with homology to telomeric DNA binding proteins

An autoantibody produced by a hybridoma derived from a viable motheaten mouse was isolated and found to have moderately high binding affinity for nucleic acids and specific preference for quadruplex DNAs. Polymerase chain reaction primers were designed to link the cloned parental antibody variable region fragments together in a subcloning vector. This single-chain variable fragment construct was then subcloned into the T7 promoter-driven expression vector pET22b(+). The construct contains (N- to C-terminal) a pelB leader sequence, variable heavy chain, glycine-serine polylinker, variable light chain, and biotin mimic peptide "strep-tag" sequence (pelB-VH-linker-VL-strep-tag). The ca. 29 kDa protein was expressed, exported to the periplasmic space of NovaBlue (DE) Escherichia coli, and purified by streptavidin affinity chromatography by binding the fused strep-tag peptide. The specificity of the purified single-chain variable fragment (scFv) for quadruplex and duplex DNAs was evaluated by a radioimmunofilterbinding assay. It retained about 10-fold higher affinity for quadruplexes relative to duplex DNA, a reduction of ca. 4-fold from the relative preferences of the parent IgG. The complementary-determining regions contain sequences that are homologous to or conservatively divergent from the key DNA-binding helix-turn-helix-forming motifs of Myb/RAP1 family telomeric DNA-binding proteins (1-3). The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.     

The ribosomal S16 protein of Escherichia coli displaying a DNA-nicking activity binds to cruciform DNA

We have recently shown that the ribosomal S16 protein of Escherichia coli is a magnesium-dependent DNase which introduces nicks into supercoiled DNA molecules [Oberto, J., Bonnefoy, E., Mouray, E., Pellegrini, O., Wikstrom, P. M. & Rouvière-Yaniv, J. (1996) Mol. Microbiol. 19, 1319-1330]. In this work we analysed the DNA-binding and DNA-nicking properties of S16 using two different approaches. Gel-retardation assays showed that S16 is a structure-specific DNA-binding protein displaying a preferential binding for cruciform DNA structures. This specific binding to cruciform DNA was further investigated using a supercoiled plasmid carrying the origin of replication of E. coli (oriC) which is an (A+T)-rich DNA region with abundant palindromic sequences susceptible of forming cruciform-like structures in vivo. We show that the nicks introduced by S16 in oriC are not randomly positioned but are precisely localised near such palindromic sequences. In addition, the nicking activity of S16 appeared to be sequence dependent since the cuts introduced by S16 occurred next to an adenine, in most cases an unpaired adenine, usually followed by a GTT sequence. Overall these experiments indicate that S16 requires a cruciform-like DNA structure to bind DNA and the presence of a particular sequence in order to introduce specific single-stranded cuts into a DNA molecule.     

Definition, distribution, and use of a conserved Bovidae retroposon element sequence motif

Based on a data-base search, the sequences of 32 Bovidae retroposon elements have been compared. Two conserved areas are identified, and one of the corresponding sequences of the derived bovine consensus was used to design oligonucleotides as primer molecules for random DNA amplification of Bovidae DNA. Such a primer binding site should occur on average every 10,000 bp in the bovine genome, as suggested by a survey of published sequences. This estimate about the distribution of these possible primer binding sites was experimentally substantiated by mapping four of these primer binding sites within 40 kb of contiguous bovine DNA, carrying the heretofore undescribed bovine lactoferrin gene. Furthermore, these conserved, ubiquitous sequence motifs prove to be useful for mapping of bovine DNA.     

N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65

We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.     

Sequence specific binding of chlamydial histone H1-like protein

Chlamydia trachomatis is one of the few prokaryotic organisms known to contain proteins that bear homology to eukaryotic histone H1. Changes in macromolecular conformation of DNA mediated by the histone H1-like protein (Hc1) appear to regulate stage specific differentiation. We have developed a cross-linking immunoprecipitation protocol to examine in vivo protein-DNA interaction by immune precipitating chlamydial Hc1 cross linked to DNA. Our results strongly support the presence of sequence specific binding sites on the chlamydial plasmid and hc1 gene upstream of its open reading frame. The preferential binding sites were mapped to 520 bp BamHI-XhoI and 547 bp BamHI-DraI DNA fragments on the plasmid and hc1 respectively. Comparison of these two DNA sequences using Bestfit program has identified a 24 bp region with >75% identity that is unique to the chlamydial genome. Double-stranded DNA prepared by annealing complementary oligonucleotides corresponding to the conserved 24 bp region bind Hc1, in contrast to control sequences with similar A+T ratios. Further, Hc1 binds to DNA in a strand specific fashion, with preferential binding for only one strand. The site specific affinity to plasmid DNA was also demonstrated by atomic force microscopy data images. Binding was always followed by coiling, shrinking and aggregation of the affected DNA. Very low protein-DNA ratio was required if incubations were carried out in solution. However, if DNA was partially immobilized on mica substrate individual strands with dark foci were still visible even after the addition of excess Hc1.