RESUMO
The vast majority of eukaryotes possess two DNA recombinases: Rad51, which is ubiquitously expressed, and Dmc1, which is meiosis-specific. The evolutionary origins of this two-recombinase system remain poorly understood. Interestingly, Dmc1 can stabilize mismatch-containing base triplets, whereas Rad51 cannot. Here, we demonstrate that this difference can be attributed to three amino acids conserved only within the Dmc1 lineage of the Rad51/RecA family. Chimeric Rad51 mutants harboring Dmc1-specific amino acids gain the ability to stabilize heteroduplex DNA joints with mismatch-containing base triplets, whereas Dmc1 mutants with Rad51-specific amino acids lose this ability. Remarkably, RAD-51 from Caenorhabditis elegans, an organism without Dmc1, has acquired "Dmc1-like" amino acids. Chimeric C. elegans RAD-51 harboring "canonical" Rad51 amino acids gives rise to toxic recombination intermediates, which must be actively dismantled to permit normal meiotic progression. We propose that Dmc1 lineage-specific amino acids involved in the stabilization of heteroduplex DNA joints with mismatch-containing base triplets may contribute to normal meiotic recombination.
Assuntos
Aminoácidos/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Recombinases/química , Recombinases/metabolismo , Recombinação Genética/genética , Aminoácidos/genética , Animais , Pareamento Incorreto de Bases , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , Mutação , Rad51 Recombinase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
DNA translocases, such as RNA polymerases, inevitably collide with nucleosomes on eukaryotic chromatin. Upon these collisions, histone chaperones are suggested to facilitate nucleosome disassembly and re-assembly. In this study, by performing in vitro transcription assays and molecular simulations, we found that partial unwrapping of a nucleosome by an RNA polymerase dramatically facilitates an H2A/H2B dimer dismantling from the nucleosome by Nucleosome Assembly Protein 1 (Nap1). Furthermore, the results uncovered molecular mechanisms of Nap1 functions in which the highly acidic C-terminal flexible tails of Nap1 contribute to the H2A/H2B binding by associating with the binding interface buried and not accessible to Nap1 globular domains, supporting the penetrating fuzzy binding mechanism seemingly shared across various histone chaperones. These findings have broad implications for the mechanisms by which histone chaperones process nucleosomes upon collisions with translocases in transcription, histone recycling and nucleosomal DNA repair.
Assuntos
Histonas , Proteína 1 de Modelagem do Nucleossomo , Nucleossomos , Cromatina , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Proteína 1 de Modelagem do Nucleossomo/genética , Células Eucarióticas/metabolismoRESUMO
T cell intracellular antigen-1 (TIA-1) plays a central role in stress granule (SG) formation by self-assembly via the prion-like domain (PLD). In the TIA-1 PLD, amino acid mutations associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Welander distal myopathy (WDM), have been identified. However, how these mutations affect PLD self-assembly properties has remained elusive. In this study, we uncovered the implicit pathogenic structures caused by the mutations. NMR analysis indicated that the dynamic structures of the PLD are synergistically determined by the physicochemical properties of amino acids in units of five residues. Molecular dynamics simulations and three-dimensional electron crystallography, together with biochemical assays, revealed that the WDM mutation E384K attenuated the sticky properties, whereas the ALS mutations P362L and A381T enhanced the self-assembly by inducing ß-sheet interactions and highly condensed assembly, respectively. These results suggest that the P362L and A381T mutations increase the likelihood of irreversible amyloid fibrillization after phase-separated droplet formation, and this process may lead to pathogenicity.
Assuntos
Aminoácidos , Esclerose Lateral Amiotrófica , Príons , Agregação Patológica de Proteínas , Antígeno-1 Intracelular de Células T , Aminoácidos/química , Aminoácidos/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Miopatias Distais/genética , Miopatias Distais/metabolismo , Humanos , Mutação , Príons/química , Agregação Patológica de Proteínas/genética , Conformação Proteica em Folha beta/genética , Domínios Proteicos/genética , Antígeno-1 Intracelular de Células T/química , Antígeno-1 Intracelular de Células T/genéticaRESUMO
Translocases such as DNA/RNA polymerases, replicative helicases, and exonucleases are involved in eukaryotic DNA transcription, replication, and repair. Since eukaryotic genomic DNA wraps around histone octamers and forms nucleosomes, translocases inevitably encounter nucleosomes. A previous study has shown that a nucleosome repositions downstream when a translocase collides with the nucleosome. However, the molecular mechanism of the downstream repositioning remains unclear. In this study, we identified the lane-switch mechanism for downstream repositioning with molecular dynamics simulations and validated it with restriction enzyme digestion assays and deep sequencing assays. In this mechanism, after a translocase unwraps nucleosomal DNA up to the site proximal to the dyad, the remaining wrapped DNA switches its binding lane to that vacated by the unwrapping, and the downstream DNA rewraps, completing downstream repositioning. This mechanism may have broad implications for transcription through nucleosomes, histone recycling, and nucleosome remodeling.
Assuntos
Montagem e Desmontagem da Cromatina , Simulação de Dinâmica Molecular , Nucleossomos/química , DNA Helicases/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Nucleossomos/metabolismoRESUMO
The condensin protein complex compacts chromatin during mitosis using its DNA-loop extrusion activity. Previous studies proposed scrunching and loop-capture models as molecular mechanisms for the loop extrusion process, both of which assume the binding of double-strand (ds) DNA to the hinge domain formed at the interface of the condensin subunits Smc2 and Smc4. However, how the hinge domain contacts dsDNA has remained unknown. Here, we conducted atomic force microscopy imaging of the budding yeast condensin holo-complex and used this data as basis for coarse-grained molecular dynamics simulations to model the hinge structure in a transient open conformation. We then simulated the dsDNA binding to open and closed hinge conformations, predicting that dsDNA binds to the outside surface when closed and to the outside and inside surfaces when open. Our simulations also suggested that the hinge can close around dsDNA bound to the inside surface. Based on these simulation results, we speculate that the conformational change of the hinge domain might be essential for the dsDNA binding regulation and play roles in condensin-mediated DNA-loop extrusion.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Biologia Computacional , Simulação por Computador , DNA Fúngico/química , Microscopia de Força Atômica , Modelos Biológicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Eletricidade EstáticaRESUMO
In physiological settings, all nucleic acids motor proteins must travel along substrates that are crowded with other proteins. However, the physical basis for how motor proteins behave in these highly crowded environments remains unknown. Here, we use real-time single-molecule imaging to determine how the ATP-dependent translocase RecBCD travels along DNA occupied by tandem arrays of high-affinity DNA binding proteins. We show that RecBCD forces each protein into its nearest adjacent neighbor, causing rapid disruption of the protein-nucleic acid interaction. This mechanism is not the same way that RecBCD disrupts isolated nucleoprotein complexes on otherwise naked DNA. Instead, molecular crowding itself completely alters the mechanism by which RecBCD removes tightly bound protein obstacles from DNA.
Assuntos
Replicação do DNA/fisiologia , DNA/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Exodesoxirribonuclease V/metabolismo , Nucleoproteínas/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Microscopia de Fluorescência , Método de Monte CarloRESUMO
Dynamics of nuclear proteins in crowded chromatin has only been poorly understood. Here, we address the diffusion, target search, and structural dynamics of three proteins in a model chromatin using coarse-grained molecular simulations run on the K computer. We prepared two structures of chromatin made of 20 nucleosomes with different nucleosome densities and investigated dynamics of two transcription factors, HMGB1 and p53, and one signaling protein, ERK, embedded in the chromatin. We found fast and normal diffusion of the nuclear proteins in the low-density chromatins and slow and subdiffusional movements in the high-density chromatin. The diffusion of the largest transcription factor, p53, is slowed by high-density chromatin most markedly. The on rates and off rates for DNA binding are increased and decreased, respectively, in the high-density chromatin. To our surprise, the DNA sequence search was faster in chromatin with high nucleosome density, though the diffusion is slower. We also found that the three nuclear proteins preferred to bind on the linker DNA and the entry and exit regions of nucleosomal DNA. In addition to these regions, HMGB1 and p53 also bound to the dyad.
Assuntos
Cromatina/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , DNA/genética , DNA/metabolismo , Difusão , Modelos Moleculares , Proteínas Nucleares/química , Domínios Proteicos , TermodinâmicaRESUMO
Protein binding to DNA changes the DNA's structure, and altered DNA structure can, in turn, modulate the dynamics of protein binding. This mutual dependency is poorly understood. Here we investigated dynamic couplings among protein binding to DNA, protein sliding on DNA, and DNA bending by applying a coarse-grained simulation method to the bacterial architectural protein HU and 14 other DNA-binding proteins. First, we verified our method by showing that the simulated HU exhibits a weak preference for A/T-rich regions of DNA and a much higher affinity for gapped and nicked DNA, consistent with biochemical experiments. The high affinity was attributed to a local DNA bend, but not the specific chemical moiety of the gap/nick. The long-time dynamic analysis revealed that HU sliding is associated with the movement of the local DNA bending site. Deciphering single sliding steps, we found the coupling between HU sliding and DNA bending is akin to neither induced-fit nor population-shift; instead they moved concomitantly. This is reminiscent of a cation transfer on DNA and can be viewed as a protein version of polaron-like sliding. Interestingly, on shorter time scales, HU paused when the DNA was highly bent at the bound position and escaped from pauses once the DNA spontaneously returned to a less bent structure. The HU sliding is largely regulated by DNA bending dynamics. With 14 other proteins, we explored the generality and versatility of the dynamic coupling and found that 6 of the 15 assayed proteins exhibit the polaron-like sliding.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Proteínas de Ligação a DNA/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Conformação de Ácido Nucleico , Ligação ProteicaRESUMO
Due to hierarchic nature of biomolecular systems, their computational modeling calls for multiscale approaches, in which coarse-grained (CG) simulations are used to address long-time dynamics of large systems. Here, we review recent developments and applications of CG modeling methods, focusing on our methods primarily for proteins, DNA, and their complexes. These methods have been implemented in the CG biomolecular simulator, CafeMol. Our CG model has resolution such that â¼10 non-hydrogen atoms are grouped into one CG particle on average. For proteins, each amino acid is represented by one CG particle. For DNA, one nucleotide is simplified by three CG particles, representing sugar, phosphate, and base. The protein modeling is based on the idea that proteins have a globally funnel-like energy landscape, which is encoded in the structure-based potential energy function. We first describe two representative minimal models of proteins, called the elastic network model and the classic GoÌ model. We then present a more elaborate protein model, which extends the minimal model to incorporate sequence and context dependent local flexibility and nonlocal contacts. For DNA, we describe a model developed by de Pablo's group that was tuned to well reproduce sequence-dependent structural and thermodynamic experimental data for single- and double-stranded DNAs. Protein-DNA interactions are modeled either by the structure-based term for specific cases or by electrostatic and excluded volume terms for nonspecific cases. We also discuss the time scale mapping in CG molecular dynamics simulations. While the apparent single time step of our CGMD is about 10 times larger than that in the fully atomistic molecular dynamics for small-scale dynamics, large-scale motions can be further accelerated by two-orders of magnitude with the use of CG model and a low friction constant in Langevin dynamics. Next, we present four examples of applications. First, the classic GoÌ model was used to emulate one ATP cycle of a molecular motor, kinesin. Second, nonspecific protein-DNA binding was studied by a combination of elaborate protein and DNA models. Third, a transcription factor, p53, that contains highly fluctuating regions was simulated on two perpendicularly arranged DNA segments, addressing intersegmental transfer of p53. Fourth, we simulated structural dynamics of dinucleosomes connected by a linker DNA finding distinct types of internucleosome docking and salt-concentration-dependent compaction. Finally, we discuss many of limitations in the current approaches and future directions. Especially, more accurate electrostatic treatment and a phospholipid model that matches our CG resolutions are of immediate importance.
Assuntos
DNA/química , Simulação de Dinâmica Molecular , Proteínas/químicaRESUMO
While fast folding of small proteins has been relatively well characterized by experiments and theories, much less is known for slow folding of larger proteins, for which recent experiments suggested quite complex and rich folding behaviors. Here, we address how the energy landscape theory can be applied to these slow folding reactions. Combining the perfect-funnel approximation with a multiscale method, we first extended our previous atomic-interaction based coarse grained (AICG) model to take into account local flexibility of protein molecules. Using this model, we then investigated the energy landscapes and folding routes of two proteins with complex topologies: a multidomain protein adenylate kinase (AKE) and a knotted protein 2ouf-knot. In the AKE folding, consistent with experimental results, the kinetic free energy surface showed several substates between the fully unfolded and native states. We characterized the structural features of these substates and transitions among them, finding temperature-dependent multiroute folding. For protein 2ouf-knot, we found that the improved atomic-interaction based coarse-grained model can spontaneously tie a knot and fold the protein with a probability up to 96%. The computed folding rate of the knotted protein was much slower than that of its unknotted counterpart, in agreement with experimental findings. Similar to the AKE case, the 2ouf-knot folding exhibited several substates and transitions among them. Interestingly, we found a dead-end substate that lacks the knot, thus suggesting backtracking mechanisms.
Assuntos
Adenilato Quinase/química , Dobramento de Proteína , Adenilato Quinase/metabolismo , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , TermodinâmicaRESUMO
In eukaryotic proteins, intrinsically disordered regions (IDRs) are ubiquitous and often exist in linker regions that flank the functional domains of modular proteins, regulating their functions. For detailed structural ensemble modeling of IDRs, we propose a multiscale method for IDRs that possess significant long-range order in modular proteins and apply it to the eukaryotic transcription factor p53 as an example. First, we performed all-atom (AA) molecular dynamics (MD) simulations of the explicitly solvated p53 linker region, without experimental restraint terms, finding fractional long-range contacts within the linker. Second, we fed this AA MD ensemble into a coarse-grained (CG) model, finding an optimal set of contact potentials. The optimized CG MD simulations reproduced the contact probability map from the AA MD simulations. Finally, we performed the CG MD simulation of the tetrameric p53 fragments including the core domains, the linker, and the tetramerization domain. Using the obtained ensemble, we theoretically calculated the small angle x-ray scattering (SAXS) profile of this fragment. The obtained SAXS profile agrees well with the experiment. We also found that the long-range contacts in the p53 linker region are required to reproduce the experimental SAXS profile. The developed framework in which we calculate the long-range contact probability map from the AA MD simulation and incorporate it to the CG model can be applied to broad range of IDRs.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Proteína Supressora de Tumor p53/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
The tumor suppressor p53 is a transcription factor that searches its cognate sites on DNA. During the search, the roles and interplay of its two DNA binding domains, the folded core domain and the disordered C-terminal domain (CTD), have been controversial. Here, we performed molecular simulations of p53 at various salt concentrations finding that, at physiological salt concentration, p53 diffuses along nonspecific DNA via rotation-uncoupled sliding with its CTD, whereas the core domain repeats dissociation and association. This is in perfect agreement with a recent single molecule experiment. In the simulation of tetrameric full-length p53, two DNA binding domains both bound to nonspecific DNA in a characteristic form at low salt concentration, whereas at physiological salt concentration, only CTD kept bound to DNA and the core domain frequently hopped on DNA. Simulations of a construct that lacks the core domain (TetCD) clarified rotation-uncoupled diffusion on nonspecific DNA. At low salt concentration, the diffusion constant due to sliding was dependent on the salt concentration, which differs from the prediction of a classic theory of transcription factors. At physiological salt concentration, it was independent of the salt concentration, in harmony with experiments. Moreover, we found that the sliding via the CTD follows the helical pitch of DNA (i.e., rotation-coupled sliding) at low salt concentration while it is virtually uncoupled to the helical pitch, a hallmark of rotation-uncoupled sliding at physiological salt concentration.
Assuntos
DNA/metabolismo , Modelos Moleculares , Rotação , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Sequência de Bases , DNA/química , DNA/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
DNA mismatches are frequently generated by various intrinsic and extrinsic factors including DNA replication errors, oxygen species, ultraviolet, and ionizing radiation. These mismatches should be corrected by the mismatches repair (MMR) pathway to maintain genome integrity. In the Escherichia coli (E. coli) MMR pathway, MutS searches and recognizes a base-pair mismatch from millions of base-pairs. Once recognized, ADP bound to MutS is exchanged with ATP, which induces a conformational change in MutS. Previous single-molecule fluorescence microscopy studies have suggested that ADP-bound MutS temporarily slides along double-stranded DNA in a rotation-coupled manner to search a base-pair mismatch and so does ATP-bound MutS in a rotation-uncoupled manner. However, the detailed structural dynamics of the sliding remains unclear. In this study, we performed coarse-grained molecular dynamics simulations of the E. coli MutS bound on DNA in three different conformations: ADP-bound (MutSADP), ATP-bound open clamp ( M u t S O p e n A T P ), and ATP-bound closed clamp ( M u t S C l o s e d A T P ) conformations. In the simulations, we observed conformation-dependent diffusion of MutS along DNA. MutSADP and M u t S C l o s e d A T P diffused along DNA in a rotation-coupled manner with rare and frequent groove-crossing events, respectively. In the groove-crossing events, MutS overcame an edge of a groove and temporarily diffused in a rotation-uncoupled manner. It was also indicated that mismatch searches by M u t S O p e n A T P is inefficient in terms of mismatch checking even though it diffuses along DNA and reaches unchecked regions more rapidly than MutSADP.
RESUMO
Intrinsically disordered proteins (IDPs) are ubiquitous and play key roles in transcriptional regulations and other cellular processes. To characterize diverse structural ensembles of IDPs, combinations of NMR and computational modeling showed some promise, but they need further improvements. Here, for accurate and efficient modeling of IDPs, we propose a systematic multiscale computational method. We first perform all-atom replica-exchange molecular dynamics (MD) simulations of a few fragments selected from a target IDP. These results together with generic knowledge-based local potentials are fed into the iterative Boltzmann inversion method to obtain an accurate coarse-grained potential. Then coarse-grained MD simulations provide the IDP ensemble. We tested the new method for the disordered N-terminal domain of p53 showing that the method reproduced the residual dipolar coupling and x-ray scattering profile very accurately. Further local structure analyses revealed that, guided by all-atom MD ensemble of fragments, the p53 N-terminal domain ensemble was biased to kinked structures in the AD1 region and biased to extended conformers in a proline-rich region and these biases contributed to improvement of the reproduction of the experiments.
Assuntos
Simulação de Dinâmica Molecular , Proteína Supressora de Tumor p53/química , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Proteína Supressora de Tumor p53/metabolismo , Difração de Raios XRESUMO
To reduce the number of replicas required in the conventional replica exchange method for huge systems, recently the replica exchange with solute tempering (REST) method was proposed. Here we showed that a variant of REST realized by rescaling the force-field parameters can be performed with GROMACS 4 without changing the code. We tested the variant REST for alanine dipeptide and an N-terminal peptide from p53 confirming its performance nearly equal to the original REST.
Assuntos
Alanina/química , Algoritmos , Dipeptídeos/química , Simulação de Dinâmica Molecular , Conformação Proteica , SolubilidadeRESUMO
The electrical properties of DNA have been extensively investigated within the field of molecular electronics. Previous studies on this topic primarily focused on the transport phenomena in the static structure at thermodynamic equilibria. Consequently, the properties of higher-order structures of DNA and their structural changes associated with the design of single-molecule electronic devices have not been fully studied so far. This stems from the limitation that only extremely short DNA is available for electrical measurements, since the single-molecule conductance decreases sharply with the increase in the molecular length. Here, we report a DNA zipper configuration to form a single-molecule junction. The duplex is accommodated in a nanogap between metal electrodes in a configuration where the duplex is perpendicular to the nanogap axis. Electrical measurements reveal that the single-molecule junction of the 90-mer DNA zipper exhibits high conductance due to the delocalized π system. Moreover, we find an attractive self-restoring capability that the single-molecule junction can be repeatedly formed without full structural breakdown even after electrical failure. The DNA zipping strategy presented here provides a basis for novel designs of single-molecule junctions.
Assuntos
DNA/química , Imagem Individual de Molécula , Simulação de Dinâmica Molecular , Análise EspectralRESUMO
Condensin plays crucial roles in chromosome organization and compaction, but the mechanistic basis for its functions remains obscure. We used single-molecule imaging to demonstrate that Saccharomyces cerevisiae condensin is a molecular motor capable of adenosine triphosphate hydrolysis-dependent translocation along double-stranded DNA. Condensin's translocation activity is rapid and highly processive, with individual complexes traveling an average distance of ≥10 kilobases at a velocity of ~60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its ~50-nanometer coiled-coil subunits, indicative of a translocation mechanism that is distinct from any reported for a DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromossomos Fúngicos/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Motores Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina , Ligação Proteica , Transporte Proteico , Saccharomyces cerevisiae/genética , Imagem Individual de MoléculaRESUMO
p53 is a representative transcription factor that activates multiple target genes. To realize stimulus-dependent specificities, p53 has to recognize targets with structural variety, of which molecular mechanisms are largely unknown. Here, we conducted a series of long-time scale (totally more than 100-ms) coarse-grained molecular dynamics simulations, uncovering structure and dynamics of full-length p53 tetramer that recognizes its response element (RE). We obtained structures of a full-length p53 tetramer that binds to the RE, which is strikingly different from the structure of p53 at search. These structures are not only consistent with previous low-resolution or partial structural information, but also give access to previously unreachable detail, such as the preferential distribution of intrinsically disordered regions, the contacts between core domains, the DNA bending, and the connectivity of linker regions. We also explored how the RE variation affects the structure of the p53-RE complex. Further analysis of simulation trajectories revealed how p53 finds out the RE and how post-translational modifications affect the search mechanism.
Assuntos
Elementos de Resposta , Proteína Supressora de Tumor p53/química , Sítios de Ligação , Cinética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
DNA strand exchange plays a central role in genetic recombination across all kingdoms of life, but the physical basis for these reactions remains poorly defined. Using single-molecule imaging, we found that bacterial RecA and eukaryotic Rad51 and Dmc1 all stabilize strand exchange intermediates in precise three-nucleotide steps. Each step coincides with an energetic signature (0.3 kBT) that is conserved from bacteria to humans. Triplet recognition is strictly dependent on correct Watson-Crick pairing. Rad51, RecA, and Dmc1 can all step over mismatches, but only Dmc1 can stabilize mismatched triplets. This finding provides insight into why eukaryotes have evolved a meiosis-specific recombinase. We propose that canonical Watson-Crick base triplets serve as the fundamental unit of pairing interactions during DNA recombination.