Binding then bending: a mechanism for wrapping DNA.
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DNA, Bacterial↗
Biomedical subjects
Phoebe A Rice
Publications and source records attributed to Phoebe A Rice.
Structure-based analysis of HU-DNA binding.
HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from approximately 10-14.5 kcal/mol, representing K(d) values in the nanomolar to low picomolar range, and a maximum stabilization of at least approximately 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.
Anabaena↗
Amino acid residues in Rag1 crucial for DNA hairpin formation.
The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, using a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acid residues to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265 and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (YKEFRK) may share functional similarities with the YREK motif in IS4 family transposases.
Amino Acid Sequence↗
An oxidation-sensing mechanism is used by the global regulator MgrA in Staphylococcus aureus.
Staphylococcus aureus is a human pathogen responsible for most wound and hospital-acquired infections. The protein MgrA is both an important virulence determinant during infection and a regulator of antibiotic resistance in S. aureus. The crystal structure of the MgrA homodimer, solved at 2.86 A, indicates the presence of a unique cysteine residue located at the interface of the protein dimer. We discovered that this cysteine residue can be oxidized by various reactive oxygen species, such as hydrogen peroxide and organic hydroperoxide. Cysteine oxidation leads to dissociation of MgrA from DNA and initiation of signaling pathways that turn on antibiotic resistance in S. aureus. The oxidation-sensing mechanism is typically used by bacteria to counter challenges of reactive oxygen and nitrogen species. Our study reveals that in S. aureus, MgrA adopts a similar mechanism but uses it to globally regulate different defensive pathways.
Bacterial Proteins↗
Mechanisms of site-specific recombination.
Integration, excision, and inversion of defined DNA segments commonly occur through site-specific recombination, a process of DNA breakage and reunion that requires no DNA synthesis or high-energy cofactor. Virtually all identified site-specific recombinases fall into one of just two families, the tyrosine recombinases and the serine recombinases, named after the amino acid residue that forms a covalent protein-DNA linkage in the reaction intermediate. Their recombination mechanisms are distinctly different. Tyrosine recombinases break and rejoin single strands in pairs to form a Holliday junction intermediate. By contrast, serine recombinases cut all strands in advance of strand exchange and religation. Many natural systems of site-specific recombination impose sophisticated regulatory mechanisms on the basic recombinational process to favor one particular outcome of recombination over another (for example, excision over inversion or deletion). Details of the site-specific recombination processes have been revealed by recent structural and biochemical studies of members of both families.
Animals↗
The structure of the human AGT protein bound to DNA and its implications for damage detection.
O6-Alklyguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that protects cells from mutagenesis and toxicity arising from alkylating agents. We present an X-ray crystal structure of the wild-type human protein (hAGT) bound to double-stranded DNA with a chemically modified cytosine base. The protein binds at two different sites: one at the modified base, and the other across a sticky-ended DNA junction. The protein molecule that binds the modified cytosine base flips the base and recognizes it in its active site. The one that binds ends of neighboring DNA molecules partially flips an overhanging thymine base. This base is not inserted into the active-site pocket of the protein. These two different hAGT/DNA interactions observed in the structure suggest that hAGT may not detect DNA lesions by searching for the adduct itself, but rather for weakened and/or distorted base-pairs caused by base damage in the duplex DNA. We propose that hAGT imposes a strain on the DNA duplex and searches for DNA regions where the native structure is destabilized. The structure provides implications for pyrimidine recognition, improved inhibitor design, and a possible protein/protein interaction patch on hAGT.
Alkyl and Aryl Transferases↗
Visualizing Mu transposition: assembling the puzzle pieces.
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Bacteriophage mu↗
Resolving integral questions in site-specific recombination.
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DNA↗
Crystal structure of a Rad51 filament.
Rad51, the major eukaryotic homologous recombinase, is important for the repair of DNA damage and the maintenance of genomic diversity and stability. The active form of this DNA-dependent ATPase is a helical filament within which the search for homology and strand exchange occurs. Here we present the crystal structure of a Saccharomyces cerevisiae Rad51 filament formed by a gain-of-function mutant. This filament has a longer pitch than that seen in crystals of Rad51's prokaryotic homolog RecA, and places the ATPase site directly at a new interface between protomers. Although the filament exhibits approximate six-fold symmetry, alternate protein-protein interfaces are slightly different, implying that the functional unit of Rad51 within the filament may be a dimer. Additionally, we show that mutation of His352, which lies at this new interface, markedly disrupts DNA binding.
Adenosine Triphosphatases↗
IHF and HU: flexible architects of bent DNA.
The energetic cost of bending short segments of DNA is very high. This bending is critical for the packaging of DNA and is exploited to regulate many cellular processes. In prokaryotes, IHF and HU are key architectural proteins present at high concentrations. New protein-DNA co-crystal structures, and the adaptation of advanced biophysical and biochemical techniques have led to an improved understanding of how these proteins interact with DNA. These techniques include time-resolved synchrotron X-ray footprinting, differential scanning calorimetry, isothermal titration calorimetry and single-molecule experiments.
Amino Acid Sequence↗
Flexible DNA bending in HU-DNA cocrystal structures.
HU and IHF are members of a family of prokaryotic proteins that interact with the DNA minor groove in a sequence-specific (IHF) or non-specific (HU) manner to induce and/or stabilize DNA bending. HU plays architectural roles in replication initiation, transcription regulation and site-specific recombination, and is associated with bacterial nucleoids. Cocrystal structures of Anabaena HU bound to DNA (1P71, 1P78, 1P51) reveal that while underlying proline intercalation and asymmetric charge neutralization mechanisms of DNA bending are similar for IHF and HU, HU stabilizes different DNA bend angles ( approximately 105-140 degrees ). The two bend angles within a single HU complex are not coplanar, and the resulting dihedral angle is consistent with negative supercoiling. Comparison of HU-DNA and IHF-DNA structures suggests that sharper bending is correlated with longer DNA binding sites and smaller dihedral angles. An HU-induced bend may be better modeled as a hinge, not a rigid bend. The ability to induce or stabilize varying bend angles is consistent with HU's role as an architectural cofactor in many different systems that may require differing geometries.
Anabaena↗
Integration host factor: putting a twist on protein-DNA recognition.
Integration host factor (IHF) is a DNA-bending protein that recognizes its cognate sites through indirect readout. Previous studies have shown that binding of wild-type (WT)-IHF is disrupted by a T to A mutation at the center position of a conserved TTR motif in its binding site, and that substitution of betaGlu44 with Ala prevented IHF from discriminating between A and T at this position. We have determined the crystal structures and relative binding affinities for all combinations of WT-IHF and IHF-betaGlu44Ala bound to the WT and mutant DNAs. Comparison of these structures reveals that DNA twist plays a major role in DNA recognition by IHF, and that this geometric parameter is dependent on the dinucleotide step and not on the bound IHF variant.
Amino Acid Substitution↗
The role of the conserved Trp330 in Flp-mediated recombination. Functional and structural analysis.
The active site of Flp contains, in addition to a transdonated nucleophilic tyrosine, five other residues that are highly conserved within the lambda-integrase family of site-specific recombinases and the type IB topoisomerases. We have used site-directed mutagenesis and x-ray crystallography to investigate the roles of two such residues, Lys223 and Trp330. Our findings agree with studies on related enzymes showing the importance of Lys223 in catalysis but demonstrate that in Flp-mediated recombination the primary role of Trp330 is architectural rather than catalytic. Eliminating the hydrogen bonding potential of Trp330 by phenylalanine substitution results in surprisingly small changes in reaction rates, compared with dramatic decreases in the activities of W330A, W330H, and W330Q. The structure of a W330F mutant-DNA complex reveals an active site nearly identical to that of the wild type. The phenylalanine side chain preserves most of the van der Waals interactions Trp330 forms with the Tyr343-containing trans helix, which may be particularly important for the docking of this helix. Our studies of Trp330 provide the first detailed examination of this conserved residue in the lambda-integrase family, suggesting that the relative importance of active site residues may differ among Flp and related enzymes.
Bacterial Proteins↗
Structural plasticity of the Flp-Holliday junction complex.
The Flp recombinase, a member of the lambda integrase or tyrosine-based family of site-specific recombinases, is an interesting example of an enzyme whose catalytic activity is regulated by protein-protein contacts. It exhibits half-of-the-sites activity throughout its catalytic cycle. Flp is unique among these recombinases, in that it assembles each active site in trans through the interaction of two protein monomers within the catalytic tetramer, with isomerization of interacting pairs being essential to complete a full reaction. We report here the structure of a DNA-bound tetramer of Flpe, a variant of Flp that is more active at 37 degrees C than the wild-type recombinase. This new structure includes the first observation of a tyrosine recombinase with an invading 5'-OH poised to attack the covalent phosphotyrosine residue. Comparison with the previously determined Flp structure highlights differences in flexibility between the two types of protein-protein interfaces in the tetramer and better defines the range of conformations available to this remarkably flexible complex. These results suggest a steric occlusion model for enforcement of half-of-the-sites activity.
Bacterial Outer Membrane Proteins↗
New insight into site-specific recombination from Flp recombinase-DNA structures.
The lamba integrase, or tyrosine-based family of site-specific recombinases, plays an important role in a variety of biological processes by inserting, excising, and inverting DNA segments. Flp, encoded by the yeast 2-mum plasmid, is the best-characterized eukaryotic member of this family and is responsible for maintaining the copy number of this plasmid. Over the past several years, structural and biochemical studies have shed light on the details of a common catalytic scheme utilized by these enzymes with interesting variations under different biological contexts. The emergence of new Flp structures and solution data provides insights not only into its unique mechanism of active site assembly and activity regulation but also into the specific contributions of certain protein residues to catalysis.
Binding Sites↗