Charge neutralization and DNA bending by the Escherichia coli catabolite activator protein

We are interested in the role of asymmetric phosphate neutralization in DNA bending induced by proteins. We describe an experimental estimate of the actual electrostatic contribution of asymmetric phosphate neutralization to the bending of DNA by the Escherichia coli catabolite activator protein (CAP), a prototypical DNA-bending protein. Following assignment of putative electrostatic interactions between CAP and DNA phosphates based on X-ray crystal structures, appropriate phosphates in the CAP half-site DNA were chemically neutralized by methylphosphonate substitution. DNA shape was then evaluated using a semi-synthetic DNA electrophoretic phasing assay. Our results confirm that the unmodified CAP DNA half-site sequence is intrinsically curved by 26° in the direction enhanced in the complex with protein. In the absence of protein, neutralization of five appropriate phosphates increases DNA curvature to 32° (~23% increase), in the predicted direction. Shifting the placement of the neutralized phosphates changes the DNA shape, suggesting that sequence-directed DNA curvature can be modified by the asymmetry of phosphate neutralization. We suggest that asymmetric phosphate neutralization contributes favorably to DNA bending by CAP, but cannot account for the full DNA deformation.     

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.     

DNA binding and bending by a chloroplast-encoded HU-like protein overexpressed in Escherichia coli

The Guillardia theta chloroplast hlpA gene encodes a protein resembling bacterial histone-like protein HU. This gene was cloned and overexpressed in Escherichia coli cells, and the resulting protein product, HlpA, was purified and characterized in vitro. In addition to exhibiting a general DNA-binding activity, the chloroplast HlpA protein also strongly facilitated cyclization of a short DNA fragment in the presence of T4 DNA ligase, indicating its ability to mediate very tight DNA curvatures.     

Opening of the replication origin of Escherichia coli by DnaA protein with protein HU or IHF.

Opening of the three tandem repeats of a 13-mer in the replication origin (oriC) of Escherichia coli is a prime event in the replication in vitro of minichromosomes (Bramhill, D., and Kornberg, A. (1988) Cell 54, 915-918). DnaA, the initiator protein, requires protein HU or IHF, along with a millimolar level of ATP and negative superhelical density in the plasmid to open this region. The extent of opening, as judged by cleavage by a single-strand-specific endonuclease (i.e. P1 nuclease), correlated closely with replication of the oriC plasmid. In an initial complex, preceding opening of the 13-mers, the footprint of DnaA protein bound by ATP covered its four 9-mer recognition sequences. The footprint of the nucleotide-free form of the protein, by contrast, was more extensive and thus, less specific.     

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.     

DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.

The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3'. Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved. In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10. All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP. Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group. Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group. dam methylation has little effect on CAP.DNA complex formation. The thermodynamically defined consensus DNA site spans 28 base pairs. All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center. The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.     

IHF redistributes bound initiator protein, DnaA, on supercoiled oriC of Escherichia coli

In Escherichia coli, initiation of chromosome replication requires that DnaA binds to R boxes (9-mer repeats) in oriC, the unique chromosomal replication origin. At the time of initiation, integration host factor (IHF) also binds to a specific site in oriC. IHF stimulates open complex formation by DnaA on supercoiled oriC in cell-free replication systems, but it is unclear whether this stimulation involves specific changes in the oriC nucleoprotein complex. Using dimethylsulphate (DMS) footprinting on supercoiled oriC plasmids, we observed that IHF redistributed prebound DnaA, stimulating binding to sites R2, R3 and R5(M), as well as to three previously unidentified non-R sites with consensus sequence (A/T)G(G/C) (A/T)N(G/C)G(A/T)(A/T)(T/C)A. Redistribution was dependent on IHF binding to its cognate site and also required a functional R4 box. By reducing the DnaA level required to separate DNA strands and trigger initiation of DNA replication at each origin, IHF eliminates competition between strong and weak sites for free DnaA and enhances the precision of initiation synchrony during the cell cycle.     

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.