The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
David B Haniford - One of the best experts on this subject based on the ideXlab platform.
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enhancement and rescue of target capture in tn10 transposition by site specific modifications in target DNA
Molecular Microbiology, 2004Co-Authors: Patrick A Pribil, Simon J Wardle, David B HanifordAbstract:Summary The bacterial transposon Tn10 inserts preferentially into specific target sequences. This insertion specificity appears to be linked to the ability of target sites to adopt symmetrically positioned DNA bends after binding the transposition machinery. Target DNA Bending is thought to permit the transposase protein to make additional contacts with the target DNA, thereby stabilizing the target complex so that the joining of transposon and target DNA sequences can occur efficiently. In the current work, we have asked whether the introduction of a discontinuity in a target DNA strand, a modification that is expected to make it easier for a DNA molecule to bend, can enhance or rescue target capture under otherwise suboptimal reaction conditions. We show that either a nick or a missing phosphate specifically at the site of reaction chemistry increases the ability of various target DNAs to form the target capture complex. The result suggests that the bends in the target DNA are highly localized and include the scissile phosphates. This raises the possibility that strand transfer is mechanistically linked to target capture. We have also identified specific residues in the target DNA and in transposase that appear to play an important role in target DNA Bending.
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target DNA Bending is an important specificity determinant in target site selection in tn10 transposition
Journal of Molecular Biology, 2003Co-Authors: Patrick A Pribil, David B HanifordAbstract:The bacterial transposon Tn10 inserts preferentially into specific DNA sequences. DNA footprinting and interference studies have revealed that the Tn10-encoded transposase protein contacts a large stretch of target DNA ( approximately 24 bp) and that the target DNA structure is deformed upon incorporation into the transpososome. Target DNA deformation might contribute significantly to target site selection and thus it is of interest to further define the nature of this deformation. Circular permutation analysis was used to demonstrate that the target DNA is bent upon its incorporation into the transpososome. Two lines of evidence are presented that target DNA Bending is an important event in target site selection. First, we demonstrate a correlation between increased target site usage and an increased level of target DNA Bending. Second, transposase mutants with relaxed target specificity are shown to cause increased target DNA Bending relative to wild-type transposase. This latter observation provides new insight into how relaxed specificity may be achieved. We also show that Ca(2+) facilitates target capture by stabilizing transposase interactions with sequences immediately flanking the insertion site. Ca(2+) could, in theory, exert this effect by stabilizing bends in the target DNA.
Phoebe A Rice - One of the best experts on this subject based on the ideXlab platform.
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two step interrogation then recognition of DNA binding site by integration host factor an architectural DNA Bending protein
Nucleic Acids Research, 2018Co-Authors: Yogambigai Velmurugu, Serguei V Kuznetsov, Paula Vivas, Phoebe A Rice, Mitchell Connolly, Anjum AnsariAbstract:The dynamics and mechanism of how site-specific DNA-Bending proteins initially interrogate potential binding sites prior to recognition have remained elusive for most systems. Here we present these dynamics for Integration Host factor (IHF), a nucleoid-associated architectural protein, using a μs-resolved T-jump approach. Our studies show two distinct DNA-Bending steps during site recognition by IHF. While the faster (∼100 μs) step is unaffected by changes in DNA or protein sequence that alter affinity by >100-fold, the slower (1-10 ms) step is accelerated ∼5-fold when mismatches are introduced at DNA sites that are sharply kinked in the specific complex. The amplitudes of the fast phase increase when the specific complex is destabilized and decrease with increasing [salt], which increases specificity. Taken together, these results indicate that the fast phase is non-specific DNA Bending while the slow phase, which responds only to changes in DNA flexibility at the kink sites, is specific DNA kinking during site recognition. Notably, the timescales for the fast phase overlap with one-dimensional diffusion times measured for several proteins on DNA, suggesting that these dynamics reflect partial DNA Bending during interrogation of potential binding sites by IHF as it scans DNA.
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target DNA Bending by the mu transpososome promotes careful transposition and prevents its reversal
eLife, 2017Co-Authors: James R Fuller, Phoebe A RiceAbstract:The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA Bending on the transposition reaction. Enhancing the flexibility of the target DNA or preBending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal.
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flexible DNA Bending in hu DNA cocrystal structures
The EMBO Journal, 2003Co-Authors: Kerren K Swinger, Kathryn M Lemberg, Ying Zhang, Phoebe A RiceAbstract: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 (∼105–140°). 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.
Patrick A Pribil - One of the best experts on this subject based on the ideXlab platform.
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enhancement and rescue of target capture in tn10 transposition by site specific modifications in target DNA
Molecular Microbiology, 2004Co-Authors: Patrick A Pribil, Simon J Wardle, David B HanifordAbstract:Summary The bacterial transposon Tn10 inserts preferentially into specific target sequences. This insertion specificity appears to be linked to the ability of target sites to adopt symmetrically positioned DNA bends after binding the transposition machinery. Target DNA Bending is thought to permit the transposase protein to make additional contacts with the target DNA, thereby stabilizing the target complex so that the joining of transposon and target DNA sequences can occur efficiently. In the current work, we have asked whether the introduction of a discontinuity in a target DNA strand, a modification that is expected to make it easier for a DNA molecule to bend, can enhance or rescue target capture under otherwise suboptimal reaction conditions. We show that either a nick or a missing phosphate specifically at the site of reaction chemistry increases the ability of various target DNAs to form the target capture complex. The result suggests that the bends in the target DNA are highly localized and include the scissile phosphates. This raises the possibility that strand transfer is mechanistically linked to target capture. We have also identified specific residues in the target DNA and in transposase that appear to play an important role in target DNA Bending.
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target DNA Bending is an important specificity determinant in target site selection in tn10 transposition
Journal of Molecular Biology, 2003Co-Authors: Patrick A Pribil, David B HanifordAbstract:The bacterial transposon Tn10 inserts preferentially into specific DNA sequences. DNA footprinting and interference studies have revealed that the Tn10-encoded transposase protein contacts a large stretch of target DNA ( approximately 24 bp) and that the target DNA structure is deformed upon incorporation into the transpososome. Target DNA deformation might contribute significantly to target site selection and thus it is of interest to further define the nature of this deformation. Circular permutation analysis was used to demonstrate that the target DNA is bent upon its incorporation into the transpososome. Two lines of evidence are presented that target DNA Bending is an important event in target site selection. First, we demonstrate a correlation between increased target site usage and an increased level of target DNA Bending. Second, transposase mutants with relaxed target specificity are shown to cause increased target DNA Bending relative to wild-type transposase. This latter observation provides new insight into how relaxed specificity may be achieved. We also show that Ca(2+) facilitates target capture by stabilizing transposase interactions with sequences immediately flanking the insertion site. Ca(2+) could, in theory, exert this effect by stabilizing bends in the target DNA.
Zippora Shakked - One of the best experts on this subject based on the ideXlab platform.
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structural and energetic origins of sequence specific DNA Bending monte carlo simulations of papillomavirus e2 DNA binding sites
Structure, 2005Co-Authors: Remo Rohs, Heinz Sklenar, Zippora ShakkedAbstract:DNA Bending is an important structural feature for indirect readout in protein-DNA recognition. The binding of papillomavirus E2 transcription factors to their DNA binding sites is associated with DNA Bending, providing an attractive model system to study the origins of sequence-specific DNA Bending. The consensus E2 target is of the general form ACCGN(4)CGGT with a variable four base pair region. We applied a new all-atom Monte Carlo (MC) algorithm that combines effective sampling with fast conformational equilibration. The resulting MC ensembles resemble the corresponding high-resolution crystal structures very well. Distinct Bending is observed for the E2-DNA binding site with a central AATT linker in contrast to an essentially straight DNA with a central ACGT linker. Contributions of specific base pair steps to the overall Bending are shown in terms of local structural parameters. The analysis of conformational substates provides new insights into the energetic origins of intrinsic DNA Bending.
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DNA Bending by an adenine thymine tract and its role in gene regulation
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Jenny Hizver, Haim Rozenberg, Felix Frolow, Dov Rabinovich, Zippora ShakkedAbstract:To gain insight into the structural basis of DNA Bending by adenine-thymine tracts (A-tracts) and their role in DNA recognition by gene-regulatory proteins, we have determined the crystal structure of the high-affinity DNA target of the cancer-associated human papillomavirus E2 protein. The three independent B-DNA molecules of the crystal structure determined at 2.2-A resolution are examples of A-tract-containing helices where the global direction and magnitude of curvature are in accord with solution data, thereby providing insights, at the base pair level, into the mechanism of DNA Bending by such sequence motifs. A comparative analysis of E2-DNA conformations with respect to other structural and biochemical studies demonstrates that (i) the A-tract structure of the core region, which is not contacted by the protein, is critical for the formation of the high-affinity sequence-specific protein-DNA complex, and (ii) differential binding affinity is regulated by the intrinsic structure and deformability encoded in the base sequence of the DNA target.
Keir C Neuman - One of the best experts on this subject based on the ideXlab platform.
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direct measurement of DNA Bending by type iia topoisomerases implications for non equilibrium topology simplification
Nucleic Acids Research, 2011Co-Authors: Ashley H Hardin, Susanta K Sarkar, Yeonee Seol, Grace F Liou, Neil Osheroff, Keir C NeumanAbstract:Type IIA topoisomerases modify DNA topology by passing one segment of duplex DNA (transfer or T–segment) through a transient double-strand break in a second segment of DNA (gate or G–segment) in an ATP-dependent reaction. Type IIA topoisomerases decatenate, unknot and relax supercoiled DNA to levels below equilibrium, resulting in global topology simplification. The mechanism underlying this non-equilibrium topology simplification remains speculative. The bend angle model postulates that non-equilibrium topology simplification scales with the bend angle imposed on the G–segment DNA by the binding of a type IIA topoisomerase. To test this bend angle model, we used atomic force microscopy and single-molecule Forster resonance energy transfer to measure the extent of Bending imposed on DNA by three type IIA topoisomerases that span the range of topology simplification activity. We found that Escherichia coli topoisomerase IV, yeast topoisomerase II and human topoisomerase IIα each bend DNA to a similar degree. These data suggest that DNA Bending is not the sole determinant of non-equilibrium topology simplification. Rather, they suggest a fundamental and conserved role for DNA Bending in the enzymatic cycle of type IIA topoisomerases.