RESUMEN
Mammalian mitotic chromosome morphogenesis was analyzed by 4D live-cell and snapshot deconvolution fluorescence imaging. Prophase chromosomes, whose organization was previously unknown, are revealed to comprise co-oriented sister linear loop arrays displayed along a single, peripheral, regularly kinked topoisomerase II/cohesin/condensin II axis. Thereafter, rather than smooth, progressive compaction as generally envisioned, progression to metaphase is a discontinuous process involving chromosome expansion as well as compaction. At late prophase, dependent on topoisomerase II and with concomitant cohesin release, chromosomes expand, axes split and straighten, and chromatin loops transit to a radial disposition around now-central axes. Finally, chromosomes globally compact, giving the metaphase state. These patterns are consistent with the hypothesis that the molecular events of chromosome morphogenesis are governed by accumulation and release of chromosome stress, created by chromatin compaction and expansion. Chromosome state could evolve analogously throughout the cell cycle.
Asunto(s)
Cromosomas de los Mamíferos/metabolismo , Metafase , Mitosis , Adenosina Trifosfatasas/análisis , Animales , Proteínas de Ciclo Celular/análisis , Línea Celular , Proteínas Cromosómicas no Histona/análisis , Cromosomas de los Mamíferos/química , ADN-Topoisomerasas de Tipo II/análisis , Proteínas de Unión al ADN/análisis , Ciervos , Células HeLa , Humanos , Microscopía Fluorescente , Complejos Multiproteicos/análisis , Porcinos , CohesinasRESUMEN
Visualization of living E. coli nucleoids, defined by HupA-mCherry, reveals a discrete, dynamic helical ellipsoid. Three basic features emerge. (1) Nucleoid density coalesces into longitudinal bundles, giving a stiff, low-DNA-density ellipsoid. (2) This ellipsoid is radially confined within the cell cylinder. Radial confinement gives helical shape and directs global nucleoid dynamics, including sister segregation. (3) Longitudinal density waves flux back and forth along the nucleoid, with 5%-10% of density shifting within 5 s, enhancing internal nucleoid mobility. Furthermore, sisters separate end-to-end in sequential discontinuous pulses, each elongating the nucleoid by 5%-15%. Pulses occur at 20 min intervals, at defined cell-cycle times. This progression includes sequential installation and release of programmed tethers, implying cyclic accumulation and relief of intranucleoid mechanical stress. These effects could comprise a chromosome-based cell-cycle engine. Overall, the presented results suggest a general conceptual framework for bacterial nucleoid morphogenesis and dynamics.
Asunto(s)
Cromosomas Bacterianos , Escherichia coli/citología , Escherichia coli/genética , Fenómenos Biomecánicos , Ciclo Celular , Replicación del ADN , ADN Bacteriano/fisiología , Escherichia coli/fisiología , TermodinámicaRESUMEN
Homologous recombination (HR) is a fundamental process common to all species. HR aims to faithfully repair DNA double strand breaks. HR involves the formation of nucleoprotein filaments on DNA single strands (ssDNA) resected from the break. The nucleoprotein filaments search for homologous regions in the genome and promote strand exchange with the ssDNA homologous region in an unbroken copy of the genome. HR has been the object of intensive studies for decades. Because multi-scale dynamics is a fundamental aspect of this process, studying HR is highly challenging, both experimentally and using computational approaches. Nevertheless, knowledge has built up over the years and has recently progressed at an accelerated pace, borne by increasingly focused investigations using new techniques such as single molecule approaches. Linking this knowledge to the atomic structure of the nucleoprotein filament systems and the succession of unstable, transient intermediate steps that takes place during the HR process remains a challenge; modeling retains a very strong role in bridging the gap between structures that are stable enough to be observed and in exploring transition paths between these structures. However, working on ever-changing long filament systems submitted to kinetic processes is full of pitfalls. This review presents the modeling tools that are used in such studies, their possibilities and limitations, and reviews the advances in the knowledge of the HR process that have been obtained through modeling. Notably, we will emphasize how cooperative behavior in the HR nucleoprotein filament enables modeling to produce reliable information.
Asunto(s)
Recombinación Homóloga , Rec A Recombinasas , Rec A Recombinasas/metabolismo , ADN de Cadena Simple/genética , Nucleoproteínas/genética , Roturas del ADN de Doble CadenaRESUMEN
Homologous recombination is a fundamental process in all living organisms that allows the faithful repair of DNA double strand breaks, through the exchange of DNA strands between homologous regions of the genome. Results of three decades of investigation and recent fruitful observations have unveiled key elements of the reaction mechanism, which proceeds along nucleofilaments of recombinase proteins of the RecA family. Yet, one essential aspect of homologous recombination has largely been overlooked when deciphering the mechanism: while ATP is hydrolyzed in large quantity during the process, how exactly hydrolysis influences the DNA strand exchange reaction at the structural level remains to be elucidated. In this study, we build on a previous geometrical approach that studied the RecA filament variability without bound DNA to examine the putative implication of ATP hydrolysis on the structure, position, and interactions of up to three DNA strands within the RecA nucleofilament. Simulation results on modeled intermediates in the ATP cycle bring important clues about how local distortions in the DNA strand geometries resulting from ATP hydrolysis can aid sequence recognition by promoting local melting of already formed DNA heteroduplex and transient reverse strand exchange in a weaving type of mechanism.
Asunto(s)
Adenosina Trifosfato/química , ADN de Cadena Simple/química , ADN/química , Recombinación Homóloga , Ácidos Nucleicos Heterodúplex/química , Rec A Recombinasas/química , Adenosina Trifosfato/metabolismo , Bacterias/genética , Bacterias/metabolismo , Sitios de Unión , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Hidrólisis , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Unión Proteica , Conformación Proteica , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismoRESUMEN
Bacterial recombinational repair of double-strand breaks often begins with creation of initiating 3' single-stranded DNA (ssDNA) tails on each side of a double-strand break (DSB). Importantly, if the RecBCD pathway is followed, RecBCD creates a gap between the sequences at 3' ends of the initiating strands. The gap flanks the DSB and extends at least to the nearest Chi site on each strand. Once the initiating strands form ssDNA-RecA filaments, each ssDNA-RecA filament searches for homologous double-stranded DNA (dsDNA) to use as a template for the DNA synthesis needed to fill the gap created by RecBCD. Our experimental results show that the DNA synthesis requires formation of a heteroduplex dsDNA that pairs >20 contiguous bases in the initiating strand with sequence matched bases in a strand from the original dsDNA. To trigger synthesis, the heteroduplex must be near the 3' end of the initiating strand. Those experimentally determined requirements for synthesis combined with the Chi site dependence of the function of RecBCD and the distribution of Chi sites in bacterial genomes could allow the RecBCD pathway to avoid some genomic rearrangements arising from directly induced DSBs; however, the same three factors could promote other rearrangements.
Asunto(s)
ADN/genética , Exodesoxirribonucleasa V/genética , Genoma Bacteriano/genética , Secuencia de Bases , ADN/biosíntesis , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Replicación del ADN/genética , ADN de Cadena Simple/genética , Escherichia coli/genética , Ácidos Nucleicos Heterodúplex/genética , Rec A Recombinasas/genética , Recombinación GenéticaRESUMEN
DNA recombination resulting from RecA-mediated strand exchange aided by RecBCD proteins often enables accurate repair of DNA double-strand breaks. However, the process of recombinational repair between short DNA regions of accidental similarity can lead to fatal genomic rearrangements. Previous studies have probed how effectively RecA discriminates against interactions involving a short similar sequence that is embedded in otherwise dissimilar sequences but have not yielded fully conclusive results. Here, we present results of in vitro experiments with fluorescent probes strategically located on the interacting DNA fragments used for recombination. Our findings suggest that DNA synthesis increases the stability of the recombination products. Fluorescence measurements can also probe the homology dependence of the extension of invading DNA strands in D-loops formed by RecA-mediated strand exchange. We examined the slow extension of the invading strand in a D-loop by DNA polymerase (Pol) IV and the more rapid extension by DNA polymerase LF-Bsu We found that when DNA Pol IV extends the invading strand in a D-loop formed by RecA-mediated strand exchange, the extension afforded by 82 bp of homology is significantly longer than the extension on 50 bp of homology. In contrast, the extension of the invading strand in D-loops by DNA LF-Bsu Pol is similar for intermediates with ≥50 bp of homology. These results suggest that fatal genomic rearrangements due to the recombination of small regions of accidental homology may be reduced if RecA-mediated strand exchange is immediately followed by DNA synthesis by a slow polymerase.
Asunto(s)
ADN Bacteriano/química , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Recombinación Homóloga , Rec A Recombinasas/química , Sondas de ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismoRESUMEN
The evolutionarily conserved Escherichia coli translesion DNA polymerase IV (DinB) is one of three enzymes that can bypass potentially deadly DNA lesions on the template strand during DNA replication. Remarkably, however, DinB is the only known translesion DNA polymerase active in RecA-mediated strand exchange during error-prone double-strand break repair. In this process, a single-stranded DNA (ssDNA)-RecA nucleoprotein filament invades homologous dsDNA, pairing the ssDNA with the complementary strand in the dsDNA. When exchange reaches the 3' end of the ssDNA, a DNA polymerase can add nucleotides onto the end, using one strand of dsDNA as a template and displacing the other. It is unknown what makes DinB uniquely capable of participating in this reaction. To explore this topic, we performed molecular modeling of DinB's interactions with the RecA filament during strand exchange, identifying key contacts made with residues in the DinB fingers domain. These residues are highly conserved in DinB, but not in other translesion DNA polymerases. Using a novel FRET-based assay, we found that DinB variants with mutations in these conserved residues are less effective at stabilizing RecA-mediated strand exchange than native DinB. Furthermore, these variants are specifically deficient in strand displacement in the absence of RecA filament. We propose that the amino acid patch of highly conserved residues in DinB-like proteins provides a mechanistic explanation for DinB's function in strand exchange and improves our understanding of recombination by providing evidence that RecA plays a role in facilitating DinB's activity during strand exchange.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Bacteriano/química , ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Rec A Recombinasas/química , ADN Bacteriano/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Rec A Recombinasas/metabolismoRESUMEN
Long non-coding RNAs (lncRNAs) are prominently associated with chromosomes in an ever-increasing diversity of roles. To provide further insight into the potential nature of these associations, we have explored, for the first time, the interaction of long single-stranded (ss) RNAs with cognate homologous double-stranded (ds) DNA in vitro Using magnetic tweezers, we measured the effects of ssRNA on force extension curves for dsDNA. We observe that the presence of ssRNA impedes the extension of dsDNA, specifically at low forces, dependent on homology between the RNA and DNA species, and dependent on ssRNA lengths (≥1 kb). The observed effect also depends on the concentration of ssRNA and is abolished by overstretching of the dsDNA. These findings show that significant homologous contacts can occur between long ssRNA and dsDNA in the absence of protein and that these contacts alter the mechanical properties of the dsDNA. We propose that long ssRNA interacts paranemically with long dsDNA via periodic short homologous interactions, e.g. mediated by RNA/DNA triplex-formation, and that dsDNA extension is impeded by formation of RNA secondary structure in the intervening unbound regions. Analogous interactions in vivo would permit lncRNAs to mediate the juxtaposition of two or more DNA regions on the same or different chromosomes.
Asunto(s)
ADN/química , ADN/genética , ARN/química , ARN/genética , Emparejamiento Base , Fenómenos Mecánicos , Conformación de Ácido Nucleico , Homología de SecuenciaRESUMEN
During DNA recombination and repair, RecA family proteins must promote rapid joining of homologous DNA. Repeated sequences with >100 base pair lengths occupy more than 1% of bacterial genomes; however, commitment to strand exchange was believed to occur after testing â¼20-30 bp. If that were true, pairings between different copies of long repeated sequences would usually become irreversible. Our experiments reveal that in the presence of ATP hydrolysis even 75 bp sequence-matched strand exchange products remain quite reversible. Experiments also indicate that when ATP hydrolysis is present, flanking heterologous dsDNA regions increase the reversibility of sequence matched strand exchange products with lengths up to â¼75 bp. Results of molecular dynamics simulations provide insight into how ATP hydrolysis destabilizes strand exchange products. These results inspired a model that shows how pairings between long repeated sequences could be efficiently rejected even though most homologous pairings form irreversible products.
Asunto(s)
Adenosina Trifosfato/metabolismo , Emparejamiento Base , ADN Bacteriano/metabolismo , Reparación del ADN por Recombinación , Secuencias Repetitivas de Ácidos Nucleicos/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN de Cadena Simple/genética , Hidrólisis , Modelos Genéticos , Conformación de Ácido Nucleico , Rec A Recombinasas/metabolismoRESUMEN
Self-organization in the cell relies on the rapid and specific binding of molecules to their cognate targets. Correct bindings must be stable enough to promote the desired function even in the crowded and fluctuating cellular environment. In systems with many nearly matched targets, rapid and stringent formation of stable products is challenging. Mechanisms that overcome this challenge have been previously proposed, including separating the process into multiple stages; however, how particular in vivo systems overcome the challenge remains unclear. Here we consider a kinetic system, inspired by homology dependent pairing between double stranded DNA in bacteria. By considering a simplified tractable model, we identify different homology testing stages that naturally occur in the system. In particular, we first model dsDNA molecules as short rigid rods containing periodically spaced binding sites. The interaction begins when the centers of two rods collide at a random angle. For most collision angles, the interaction energy is weak because only a few binding sites near the collision point contribute significantly to the binding energy. We show that most incorrect pairings are rapidly rejected at this stage. In rare cases, the two rods enter a second stage by rotating into parallel alignment. While rotation increases the stability of matched and nearly matched pairings, subsequent rotational fluctuations reduce kinetic trapping. Finally, in vivo chromosome are much longer than the persistence length of dsDNA, so we extended the model to include multiple parallel collisions between long dsDNA molecules, and find that those additional interactions can greatly accelerate the searching.
Asunto(s)
Emparejamiento Base/genética , ADN/química , ADN/genética , Modelos Químicos , Modelos Moleculares , Homología de Secuencia de Ácido Nucleico , Sitios de Unión , Simulación por Computador , Reproducibilidad de los ResultadosRESUMEN
RecA family proteins include RecA, Rad51, and Dmc1. These recombinases are responsible for homology search and strand exchange. Homology search and strand exchange occur during double-strand break repair and in eukaryotes during meiotic recombination. In bacteria, homology search begins when RecA binds an initiating single-stranded DNA (ssDNA) in the primary DNA-binding site to form the presynaptic filament. The filament is a right-handed helix, where the initiating strand is bound deep within the filament. Once the presynaptic filament is formed, it interrogates nearby double-stranded DNA (dsDNA) to find a homologous sequence; therefore, we provide a detailed discussion of structural features of the presynaptic filament that play important functional roles. The discussion includes many diagrams showing multiple filament turns. These diagrams illustrate interactions that are not evident in single turn structures. The first dsDNA interactions with the presynaptic filament are insensitive to mismatches. The mismatch insensitive interactions lead to dsDNA deformation that triggers a homology testing process governed by kinetics. The first homology test involves â¼8 bases. Almost all interactions are rejected by this initial rapid test, leading to a new cycle of homology testing. Interactions that pass the initial rapid test proceed to a slower testing stage. That slower stage induces nonhomologous dsDNA to reverse strand exchange and begin a new cycle of homology testing. In contrast, homologous dsDNA continues to extend the heteroduplex strand-exchange product until ATP hydrolysis makes strand exchange irreversible.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , ADN/genética , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo , Animales , ADN/química , ADN/metabolismo , Recombinación Homóloga , Humanos , Meiosis , Modelos Moleculares , Conformación ProteicaRESUMEN
RecA family proteins are responsible for homology search and strand exchange. In bacteria, homology search begins after RecA binds an initiating single-stranded DNA (ssDNA) in the primary DNA-binding site, forming the presynaptic filament. Once the filament is formed, it interrogates double-stranded DNA (dsDNA). During the interrogation, bases in the dsDNA attempt to form Watson-Crick bonds with the corresponding bases in the initiating strand. Mismatch dependent instability in the base pairing in the heteroduplex strand exchange product could provide stringent recognition; however, we present experimental and theoretical results suggesting that the heteroduplex stability is insensitive to mismatches. We also present data suggesting that an initial homology test of 8 contiguous bases rejects most interactions containing more than 1/8 mismatches without forming a detectable 20 bp product. We propose that, in vivo, the sparsity of accidental sequence matches allows an initial 8 bp test to rapidly reject almost all non-homologous sequences. We speculate that once the initial test is passed, the mismatch insensitive binding in the heteroduplex allows short mismatched regions to be incorporated in otherwise homologous strand exchange products even though sequences with less homology are eventually rejected.
Asunto(s)
Disparidad de Par Base , ADN Forma B/metabolismo , ADN de Cadena Simple/metabolismo , Rec A Recombinasas/metabolismo , ADN Forma B/química , Transferencia Resonante de Energía de Fluorescencia , Cinética , Simulación de Dinámica Molecular , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/metabolismo , Unión Proteica , Rec A Recombinasas/química , Homología de Secuencia de Ácido NucleicoRESUMEN
RecA protein is the prototypical recombinase. Members of the recombinase family can accurately repair double strand breaks in DNA. They also provide crucial links between pairs of sister chromatids in eukaryotic meiosis. A very broad outline of how these proteins align homologous sequences and promote DNA strand exchange has long been known, as are the crystal structures of the RecA-DNA pre- and postsynaptic complexes; however, little is known about the homology searching conformations and the details of how DNA in bacterial genomes is rapidly searched until homologous alignment is achieved. By integrating a physical model of recognition to new modeling work based on docking exploration and molecular dynamics simulation, we present a detailed structure/function model of homology recognition that reconciles extremely quick searching with the efficient and stringent formation of stable strand exchange products and which is consistent with a vast body of previously unexplained experimental results.
Asunto(s)
ADN Forma B/química , ADN de Cadena Simple/química , Recombinación Homóloga , Rec A Recombinasas/química , Emparejamiento Base , ADN Forma B/metabolismo , ADN de Cadena Simple/metabolismo , Simulación de Dinámica Molecular , Unión Proteica , Rec A Recombinasas/metabolismoRESUMEN
RecA and Rad51 proteins play an important role in DNA repair and homologous recombination. For RecA, X-ray structure information and single molecule force experiments have indicated that the differential extension between the complementary strand and its Watson-Crick pairing partners promotes the rapid unbinding of non-homologous dsDNA and drives strand exchange forward for homologous dsDNA. In this work we find that both effects are also present in Rad51 protein. In particular, pulling on the opposite termini (3' and 5') of one of the two DNA strands in a dsDNA molecule allows dsDNA to extend along non-homologous Rad51-ssDNA filaments and remain stably bound in the extended state, but pulling on the 3'5' ends of the complementary strand reduces the strand-exchange rate for homologous filaments. Thus, the results suggest that differential extension is also present in dsDNA bound to Rad51. The differential extension promotes rapid recognition by driving the swift unbinding of dsDNA from non-homologous Rad51-ssDNA filaments, while at the same time, reducing base pair tension due to the transfer of the Watson-Crick pairing of the complementary strand bases from the highly extended outgoing strand to the slightly less extended incoming strand, which drives strand exchange forward.
Asunto(s)
ADN/metabolismo , Recombinación Homóloga , Recombinasa Rad51/metabolismo , ADN de Cadena Simple/metabolismo , HumanosRESUMEN
RecA-family proteins mediate homologous recombination and recombinational DNA repair through homology search and strand exchange. Initially, the protein forms a filament with the incoming single-stranded DNA (ssDNA) bound in site I. The RecA-ssDNA filament then binds double-stranded DNA (dsDNA) in site II. Non-homologous dsDNA rapidly unbinds, whereas homologous dsDNA undergoes strand exchange yielding heteroduplex dsDNA in site I and the leftover outgoing strand in site II. We show that applying force to the ends of the complementary strand significantly retards strand exchange, whereas applying the same force to the outgoing strand does not. We also show that crystallographically determined binding site locations require an intermediate structure in addition to the initial and final structures. Furthermore, we demonstrate that the characteristic dsDNA extension rates due to strand exchange and free RecA binding are the same, suggesting that relocation of the complementary strand from its position in the intermediate structure to its position in the final structure limits both rates. Finally, we propose that homology recognition is governed by transitions to and from the intermediate structure, where the transitions depend on differential extension in the dsDNA. This differential extension drives strand exchange forward for homologs and increases the free energy penalty for strand exchange of non-homologs.
Asunto(s)
ADN/química , ADN/metabolismo , Recombinación Homóloga , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo , ADN de Cadena Simple/metabolismoRESUMEN
A RecA-single-stranded DNA (RecA-ssDNA) filament searches a genome for sequence homology by rapidly binding and unbinding double-stranded DNA (dsDNA) until homology is found. We demonstrate that pulling on the opposite termini (3' and 5') of one of the two DNA strands in a dsDNA molecule stabilizes the normally unstable binding of that dsDNA to non-homologous RecA-ssDNA filaments, whereas pulling on the two 3', the two 5', or all four termini does not. We propose that the 'outgoing' strand in the dsDNA is extended by strong DNA-protein contacts, whereas the 'complementary' strand is extended by the tension on the base pairs that connect the 'complementary' strand to the 'outgoing' strand. The stress resulting from different levels of tension on its constitutive strands causes rapid dsDNA unbinding unless sufficient homology is present.
Asunto(s)
ADN/química , Rec A Recombinasas/metabolismo , Estrés Mecánico , ADN/metabolismo , ADN de Cadena Simple/metabolismo , Rotación , Homología de Secuencia de Ácido NucleicoRESUMEN
RecA is a key protein in homologous recombination. During recombination, one single-stranded DNA (ssDNA) bound to site I in RecA exchanges Watson-Crick pairing with a sequence-matched ssDNA that was part of a double-stranded DNA molecule (dsDNA) bound to site II in RecA. After strand exchange, heteroduplex dsDNA is bound to site I. In vivo, direct polymerization of RecA on dsDNA through site I does not occur, though it does in vitro. The mechanisms underlying the difference have been unclear. We use single-molecule experiments to decouple the two steps involved in polymerization: nucleation and elongation. We find that elongation is governed by a fundamental clock that is insensitive to force and RecA concentration from 0.2 and 6 µM, though rates depend on ionic conditions. Thus, we can probe nucleation site stability by creating nucleation sites at high force and then measuring elongation as a function of applied force. We find that in the presence of ATP hydrolysis a minimum force is required for polymerization. The minimum force decreases with increasing RecA or ATP concentrations. We propose that force reduces the off-rate for nucleation site binding and that nucleation site stability is the stringency factor that prevents in vivo polymerization.
Asunto(s)
ADN/metabolismo , Rec A Recombinasas/metabolismo , Adenosina Trifosfato/metabolismo , ADN/química , Hidrólisis , Polimerizacion , Rec A Recombinasas/químicaRESUMEN
The RecA protein is an ATPase that mediates recombination via strand exchange. In strand exchange a single-stranded DNA (ssDNA) bound to RecA binding site I in a RecA/ssDNA filament pairs with one strand of a double-stranded DNA (dsDNA) and forms heteroduplex dsDNA in site I if homology is encountered. Long sequences are exchanged in a dynamic process in which initially unbound dsDNA binds to the leading end of a RecA/ssDNA filament, while heteroduplex dsDNA unbinds from the lagging end via ATP hydrolysis. ATP hydrolysis is required to convert the active RecA conformation, which cannot unbind, to the inactive conformation, which can unbind. If dsDNA extension due to RecA binding increases the dsDNA tension, then RecA unbinding must decrease tension. We show that in the presence of ATP hydrolysis decreases in tension induce decreases in length whereas in the absence of hydrolysis, changes in tension have no systematic effect. These results suggest that decreases in force enhance dissociation by promoting transitions from the active to the inactive RecA conformation. In contrast, increases in tension reduce dissociation. Thus, the changes in tension inherent to strand exchange may couple with ATP hydrolysis to increase the directionality and stringency of strand exchange.
Asunto(s)
ADN/química , Rec A Recombinasas/química , Adenosina Trifosfato/metabolismo , Tampones (Química) , ADN/metabolismo , Conformación Proteica , Rec A Recombinasas/metabolismoRESUMEN
In E. coli, double strand breaks (DSBs) are resected and loaded with RecA protein. The genome is then rapidly searched for a sequence that is homologous to the DNA flanking the DSB. Mismatches in homologous partners are rare, suggesting that RecA should rapidly reject mismatched recombination products; however, this is not the case. Decades of work have shown that long lasting recombination products can include many mismatches. In this work, we show that in vitro RecA forms readily observable recombination products when 16% of the bases in the product are mismatched. We also consider various theoretical models of mismatch-tolerant homology testing. The models test homology by comparing the sequences of Ltest bases in two single-stranded DNAs (ssDNA) from the same genome. If the two sequences pass the homology test, the pairing between the two ssDNA becomes permanent. Stringency is the fraction of permanent pairings that join ssDNA from the same positions in the genome. We applied the models to both randomly generated genomes and bacterial genomes. For both randomly generated genomes and bacterial genomes, the models show that if no mismatches are accepted stringency is â¼ 99% when Ltest = 14 bp. For randomly generated genomes, stringency decreases with increasing mismatch tolerance, and stringency improves with increasing Ltest. In contrast, in bacterial genomes when Ltest â¼ 75 bp, stringency is â¼ 99% for both mismatch-intolerant and mismatch-tolerant homology testing. Furthermore, increasing Ltest does not improve stringency because most incorrect pairings join different copies of repeats. In sum, for bacterial genomes highly mismatch tolerant homology testing of 75 bp provides the same stringency as homology testing that rejects all mismatches and testing more than â¼75 base pairs is not useful. Interestingly, in vivo commitment to recombination typically requires homology testing of â¼ 75 bp, consistent with highly mismatch intolerant testing.
Asunto(s)
ADN , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Emparejamiento Base , ADN de Cadena Simple/genética , Recombinación GenéticaRESUMEN
This paper describes an empirical model of polymer dynamics, based on the agitation of millimeter-sized polymeric beads. Although the interactions between the particles in the macroscopic model and those between the monomers of molecular-scale polymers are fundamentally different, both systems follow the Worm-Like Chain theory.