Role of architectural elements in combinatorial regulation of initiation of DNA replication in Escherichia coli

Bending of DNA is a prerequisite of site-specific recombination and gene expression in many regulatory systems involving the assembly of specific nucleoprotein complexes. We have investigated how the uniquely clustered Dam methylase sites, GATCs, in the origin of Escherichia coli replication ( oriC  ) and their methylation status modulate the geometry of oriC and its interaction with architectural proteins, such as integration host factor (IHF), factor for inversion stimulation (Fis) and DnaA initiator protein. We note that 3 of the 11 GATC sites at oriC are strategically positioned within the IHF protected region. Methylation of the GATCs enhances IHF binding and alters the IHF-induced bend at oriC . GATC motifs also contribute to intrinsic DNA curvature at oriC and the degree of bending is modulated by methylation. The IHF-induced bend at oriC is further modified by Fis protein and IHF affinity for its binding site may be impaired by protein(s) binding to GATCs within the IHF site. Thus, GATC sites at oriC affect the DNA conformation and GATCs, in conjunction with the protein-induced bends, are critical cis -acting elements in specifying proper juxtapositioning of initiation factors in the early steps of DNA replication.     

DNA bending of pSC101 ori induced by Rep,a replication initiator protein and IHF

Purified Rep (or RepA) protein, a replication initiator of plasmid pSC101, is present almost solely in the dimer form, and its binding activity for the directly repeated sequences (iterons) in the replication origin (ori) is very low. When Rep protein was treated with guanidine hydrochloride followed by renaturation, it was shown to bind to the iterons with very high efficiency. A gel shift experiment suggested that guanidine-treated Rep bound to iterons as a monomer form. The Rep monomer bound noncooperatively to the three iterons and induced bending of the DNA helix axis in the same direction (about 100 degrees ). The configuration of the IHF box that is a binding site of another DNA bending protein IHF, the three iterons and an AT-rich region between these sequences was important for efficient bending of the ori region. Furthermore, a mutant Rep protein (Rep(IHF)) which can support the plasmid replication in IHF-deficient host cells was purified, and it was found that affinity of the Rep(IHF) monomer for iterons was similar to that of wild-type Rep and bent DNA only 14 degrees more strongly than did the wild-type Rep. Rep(IHF)-dependent plasmid replication, however, required both enhancer regions, par and IR-1, in addition to "core ori" as a minimal essential ori, whereas only one of these two enhancers was necessary for wild-type Rep-dependent replication. How Rep(IHF) can support plasmid replication in the absence of IHF is discussed.     

Mapping the transition state for DNA bending by IHF

How DNA-bending proteins recognize their specific sites on DNA remains elusive, particularly for proteins that use indirect readout, which relies on sequence-dependent variations in DNA flexibility/bendability. The question remains as to whether the protein bends the DNA (protein-induced bending) or, alternatively, "prebent" DNA conformations are thermally accessible, which the protein captures to form the specific complex (conformational capture). To distinguish between these mechanisms requires characterization of reaction intermediates and, in particular, snapshots of the transition state along the recognition pathway. We present such a snapshot, from measurements of DNA bending dynamics in complex with Escherichia coli integration host factor (IHF), an architectural protein that bends specific sites on λ-DNA in a U-turn by creating two sharp kinks in DNA. Fluorescence resonance energy transfer measurements in response to laser temperature-jump perturbation monitor DNA bending. We find that nicks or mismatches that enhance DNA flexibility at the site of the kinks show 3- to 4-fold increase in DNA bending rates that reflect a 4- to 11-fold increase in binding affinities, while sequence modifications away from the kink sites, as well as mutations in IHF designed to destabilize the complex, have negligible effect on DNA bending rates despite >250-fold decrease in binding affinities. These results support the scenario that the bottleneck in the recognition step for IHF is spontaneous kinking of cognate DNA to adopt a partially prebent conformation and point to conformational capture as the underlying mechanism of initial recognition, with additional protein-induced bending occurring after the transition state.     

In vitro interactions of the histone-like protein IHF with the algD promoter, a critical site for control of mucoidy in Pseudomonas aeruginosa.

The ability of the histone-like element Integration Host Factor (IHF) to interact with the algD promoter was investigated. IHF from Escherichia coli was found to bind to the algD promoter and to form multiple protein-DNA complexes in gel mobility shift DNA binding assay. The highest affinity binding site for IHF was mapped by DNaseI footprinting analysis. This site spanned nucleotides -50 to -85 relative to the algD mRNA start site and overlapped a sequence matching the IHF consensus sequence WATCAANNNNTTR in 12 out of 13 base pairs. Previous studies have shown that deletion of sequences including a portion of this site adversely affects algD promoter activity. IHF binding to the algD promoter induced DNA bending. Western blot analysis with antibodies against E. coli IHF detected a cross-reactive protein of a similar molecular mass in Pseudomonas aeruginosa, suggesting the presence of an analogous factor in this organism.     

Variation of the intercalating proline in artificial peptides mimicking the DNA binding and bending IHF protein

The integration host factor (IHF) is a protein which sequence specifically induces a bend of double-stranded DNA by more than 160°. Based on IHF as lead structure, a peptide mimic was introduced resembling the positively charged body of the protein by a lysine dendrimer and the minor groove recognition loop by a cyclopeptide. The proline located close to the tip of the recognition loop intercalates between the base pair plane. It was modified in order to evaluate the influence of the side chain residue with respect to size (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), aromaticity (phenylalanine), conformation of the five-membered ring [(4R)-fluoroproline, (4S)-fluoroproline, 3,4-dehydroproline], and the peptide backbone conformation (α-methylproline) on binding dsDNA and bending the double strand. Binding and bending studies were carried out by fluorescence resonance energy transfer experiments and gel electrophoresis using DNA sequences prepared by PCR with the IHF binding site in central or terminal position. Whereas aromatic residues and α-methylproline were not tolerated as proline substitute, incorporation of (4S)-fluoroproline and 3,4-dehydroproline provided enhanced binding.     

In Vitro Selection of Integration Host Factor Binding Sites

Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3' domain is conserved, the 5' domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5' domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5' binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5' binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.     

Examining the contribution of a dA+dT element to the conformation of Escherichia coli integration host factor-DNA complexes.

DNA binding proteins that induce structural changes in DNA are common in both prokaryotes and eukaryotes. Integration host factor (IHF) is a multi-functional DNA binding and bending protein of Escherichia coli that can mediate protein-protein and protein-DNA interactions by bending DNA. Previously we have shown that the presence of a dA+dT element 5'-proximal to an IHF consensus sequence can affect the binding of IHF to a particular site. In this study the contribution of various sequence elements to the formation of IHF-DNA complexes was examined. We show that IHF bends DNA more when it binds to a site containing a dA+dT element upstream of its core consensus element than to a site lacking a dA+dT element. We demonstrate that IHF can be specifically crosslinked to DNA with binding sites either containing or lacking this dA+dT element. These results indicate the importance of flanking DNA and a dA+dT element in the binding and bending of a site by IHF.     

Genetic analysis of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda.

cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, consists of three binding sites (called R3, R2 and R1) for gpNu1, the small subunit of terminase; and I1, a binding site for integration host factor (IHF), the DNA bending protein of Escherichia coli. cosB is located between cosN, the site where terminase introduces staggered nicks to generate cohesive ends, and the Nu1 gene; the order of sites is: cosN-R3-I1-R2-R1-Nu1. A series of lambda mutants have been constructed that have single base-pair C-to-T transition mutations in R3, R2 and R1. A single base-pair transition mutation within any one of the gpNul binding sites renders lambda dependent upon IHF for plaque formation. lambda phage with mutations in both R2 and R3 are incapable of plaque formation even in the presence of IHF. Phages that carry DNA insertions between R1 and R2, from 7 to 20 base-pairs long, are also IHF-dependent, demonstrating the requirement for a precise spacing of gpNu1 binding sites within cosB. The IHF-dependent phenotype of a lambda mutant carrying a deletion of the R1 sequence indicates that IHF obviates the need for terminase binding to the R1 site. In contrast, a lambda mutant deleted for R2 and R1 fails to form plaques on either IHF+ or IHF- cells, indicating terminase binding of R2 is involved in suppression of R mutants by IHF. A fourth R sequence, R4, is situated on the left side of cosN; a phage with a mutant R4 sequence shows a reduced burst size on both an IHF+ and an IHF- host. The inability of the R4- mutant to be suppressed by IHF, plus the fact that R4 does not bind gpNu1, suggests R4 is not part of cosB and may play a role in DNA packaging that is distinct from that of cosB.     

Building the bacterial orisome: high‐affinity DnaA recognition plays a role in setting the conformation of oriC DNA

During assembly of the E. coli pre‐replicative complex (pre‐RC), initiator DnaA oligomers are nucleated from three widely separated high‐affinity DnaA recognition sites in oriC. Oligomer assembly is then guided by low‐affinity DnaA recognition sites, but is also regulated by a switch‐like conformational change in oriC mediated by sequential binding of two DNA bending proteins, Fis and IHF, serving as inhibitor and activator respectively. Although their recognition sites are separated by up to 90 bp, Fis represses IHF binding and weak DnaA interactions until accumulating DnaA displaces Fis from oriC. It remains unclear whether high‐affinity DnaA binding plays any role in Fis repression at a distance and it is also not known whether all high‐affinity DnaA recognition sites play an equivalent role in oligomer formation. To examine these issues, we developed origin‐selective recombineering methods to mutate E. coli chromosomal oriC. We found that, although oligomers were assembled in the absence of any individual high‐affinity DnaA binding site, loss of DnaA binding at peripheral sites eliminated Fis repression, and made binding of both Fis and IHF essential. We propose a model in which interaction of DnaA molecules at high‐affinity sites regulates oriC DNA conformation.     

Bacteriophage lambda gpNu1 and Escherichia coli IHF proteins cooperatively bind and bend viral DNA: implications for the assembly of a genome-packaging motor

Terminase enzymes are common to both prokaryotic and eukaryotic double-stranded DNA viruses and are responsible for packaging viral DNA into the confines of an empty procapsid shell. In all known cases, the holoenzymes are heteroligomers composed of a large subunit that possesses the catalytic activities required for genome packaging and a small subunit that is responsible for specific recognition of viral DNA. In bacteriophage lambda, the DNA recognition protein is gpNu1. The gpNu1 subunit interacts with multiple recognition elements within cos, the packaging initiation site in viral DNA, to site-specifically assemble the packaging machinery. Motor assembly is modulated by the Escherichia coli integration host factor protein (IHF), which binds to a consensus sequence also located within cos. On the basis of a variety of biochemical data and the recently solved NMR structure of the DNA binding domain of gpNu1, we proposed a novel DNA binding mode that predicts significant bending of duplex DNA by gpNu1 (de Beer et al. (2002) Mol. Cell 9, 981-991). We further proposed that gpNu1 and IHF cooperatively bind and bend viral DNA to regulate the assembly of the packaging motor. Here, we characterize cooperative gpNu1 and IHF binding to the cos site in lambda DNA using a quantitative electrophoretic mobility shift (EMS) assay. These studies provide direct experimental support for the long presumed cooperative assembly of gpNu1 and IHF at the cos sequence of lambda DNA. Further, circular permutation experiments demonstrate that the viral and host proteins each introduce a strong bend in cos-containing DNA, but not nonspecific DNA substrates. Thus, specific recognition of viral DNA by the packaging apparatus is mediated by both DNA sequence information and by structural alteration of the duplex. The relevance of these results with respect to the assembly of a viral DNA-packaging motor is discussed.     

The integration host factor of Escherichia coli binds to bent DNA at the origin of replication of the plasmid pSC101

The integration host factor (IHF) of Escherichia coli is necessary for maintenance of pSC101. The protein binds specifically to the replication origin of the plasmid, in the AT-rich region located immediately adjacent to the left, weak binding site for the plasmid-encoded initiator protein. DNAase I and OH- radical footprinting experiments showed that IHF protects 49 bp of the DNA at the origin region. Methylation protection analyses revealed that IHF contacts purine residues in both the major and minor grooves of the DNA. Electrophoretic analyses showed that IHF binds to bent DNA, and the protein binding further enhances the degree of DNA bending. Site-directed mutagenesis of three of the contact points not only abolished binding of the protein to the DNA but also inactivated the replication origin. Therefore, binding of IHF to the ori sequence most probably is necessary for initiation of plasmid replication.     

Efficient specific DNA binding by p53 requires both its central and C-terminal domains as revealed by studies with high-mobility group 1 protein

The nonhistone chromosomal protein high-mobility group 1 protein (HMG-1/HMGB1) can serve as an activator of p53 sequence-specific DNA binding (L. Jayaraman, N. C. Moorthy, K. G. Murthy, J. L. Manley, M. Bustin, and C. Prives, Genes Dev. 12:462-472, 1998). HMGB1 is capable of interacting with DNA in a non-sequence-specific manner and causes a significant bend in the DNA helix. Since p53 requires a significant bend in the target site, we examined whether DNA bending by HMGB1 may be involved in its enhancement of p53 sequence-specific binding. Accordingly, a 66-bp oligonucleonucleotide containing a p53 binding site was locked in a bent conformation by ligating its ends to form a microcircle. Indeed, p53 had a dramatically greater affinity for the microcircle than for the linear 66-bp DNA. Moreover, HMGB1 augmented binding to the linear DNA but not to the microcircle, suggesting that HMGB1 works by providing prebent DNA to p53. p53 contains a central core sequence-specific DNA binding region and a C-terminal region that recognizes various forms of DNA non-sequence specifically. The p53 C terminus has also been shown to serve as an autoinhibitor of core-DNA interactions. Remarkably, although the p53 C terminus inhibited p53 binding to the linear DNA, it was required for the increased affinity of p53 for the microcircle. Thus, depending on the DNA structure, the p53 C terminus can serve as a negative or a positive regulator of p53 binding to the same sequence and length of DNA. We propose that both DNA binding domains of p53 cooperate to recognize sequence and structure in genomic DNA and that HMGB1 can help to provide the optimal DNA structure for p53.     

Binding of histone H1 to DNA is described by an allosteric model

Equilibrium binding data were analyzed to characterize the interaction of the linker histone H1 degrees with unmodified T4 phage DNA. Data were cast into the Scatchard-type plot described by McGhee and von Hippel and fit to their eponymous model for nonspecific binding of ligand to DNA. The data were not fit by the simple McGhee-von Hippel model, nor fit satisfactorily by the inclusion of a cooperativity parameter. Instead, the interaction appeared to be well described by Crothers' allosteric model, in which the higher affinity of the protein for one conformational form of the DNA drives an allosteric transition of the DNA to the conformational form with higher affinity (form 2). At 214 mM Na(+), the observed affinity K for an isolated site on unmodified T4 bacteriophage DNA in the form 2 conformation is 4.5 x 10(7) M(-1). The binding constant for an isolated site on DNA in the conformation with lower affinity, form 1, appears to be about 10-fold lower. Binding affinity is dependent on ion concentration: the magnitude of K is about 10-fold higher at 14 mM (5.9 x 10(8) M(-1) for form 2 DNA) than at 214 mM Na(+) concentration.     

Specific and nonspecific interactions of integration host factor with oligo DNAs as revealed by circular dichroism spectroscopy and filter binding assay.

Binding specificity of integration host factor (IHF) to oligo DNAs has been studied by circular dichroism (CD) spectroscopy and filter binding experiment. CD difference spectra of IHF-DNA complexes demonstrated that a conformational change in DNA was induced by binding of IHF when DNA had a consensus sequence for the binding sites of IHF, but that such conformational change was not observed for consensus DNA 20 mer as well as nonconsensus DNA 45 mer. Dissociation constants for IHF-DNA complexes determined by filter binding assay showed that IHF has indeed stronger affinity to DNA with the consensus binding site than to nonconsensus DNA, but the difference in its affinity between consensus and nonconsensus DNAs was rather small, 3.4-fold. It was, therefore, concluded that the flanking regions of the consensus sequence are important for the specific binding of IHF and that its binding specificity is well characterized by the induced conformational change in DNA rather than by dissociation constants for IHF-DNA complexes.     

Bending of the bacteriophage lambda attachment site by Escherichia coli integration host factor

Escherichia coli integration host factor (IHF) is a small basic protein that is required for efficient integrative recombination of bacteriophage lambda. IHF binds specifically to sequences within attP, the site in bacteriophage lambda that undergoes recombination. It has been suggested that the binding of IHF creates bends in DNA so as to help attP condense into a compact structure that is activated for recombination. In this work we show that IHF binding to either of two sites found within attP does indeed produce bending of DNA. In contrast, the other recombination protein needed for integrative recombination, Int, does not appreciably bend the DNA to which it is bound. In agreement with the proposal that IHF bending is important for creating a condensed attP, bending by IHF persists in the presence of bound Int. Our conclusions about protein-directed bends in DNA are based on the study of the electrophoretic mobility of a set of permuted DNA fragments in the presence or absence of IHF and/or Int. To facilitate this study, we have constructed a novel vector that simplifies the generation of permuted fragments. This vector should be useful in studying the bending of other DNA sequences by specific binding proteins.