RecA finds homologous DNA by reduced dimensionality search.
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ABSTRACT:
SUBMITTER: Wiktor J
PROVIDER: S-EPMC8443446 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:During homologous recombination, RecA forms a helical filament on a single stranded (ss) DNA that searches for a homologous double stranded (ds) DNA and catalyzes the exchange of complementary base pairs to form a new heteroduplex. Using single molecule fluorescence imaging tools with high spatiotemporal resolution we characterized the encounter complex between the RecA filament and dsDNA. We present evidence in support of the 'sliding model' wherein a RecA filament diffuses along a dsDNA track. We further show that homology can be detected during sliding. Sliding occurs with a diffusion coefficient of approximately 8000 bp(2)/s allowing the filament to sample several hundred base pairs before dissociation. Modeling suggests that sliding can accelerate homology search by as much as 200 fold. Homology recognition can occur for as few as 6 nt of complementary basepairs with the recognition efficiency increasing for higher complementarity. Our data represents the first example of a DNA bound multi-protein complex which can slide along another DNA to facilitate target search.DOI:http://dx.doi.org/10.7554/eLife.00067.001.
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Project description: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.
| S-EPMC6735932 | biostudies-literature
Project description: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.
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Project description:DNA breaks can be repaired with high fidelity by homologous recombination. A ubiquitous protein that is essential for this DNA template-directed repair is RecA. After resection of broken DNA to produce single-stranded DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous to the ssDNA. This homology search is vital to recombinational DNA repair, and results in homologous pairing and exchange of DNA strands. Homologous pairing involves DNA sequence-specific target location by the RecA-ssDNA complex. Despite decades of study, the mechanism of this enigmatic search process remains unknown. RecA is a DNA-dependent ATPase, but ATP hydrolysis is not required for DNA pairing and strand exchange, eliminating active search processes. Using dual optical trapping to manipulate DNA, and single-molecule fluorescence microscopy to image DNA pairing, we demonstrate that both the three-dimensional conformational state of the dsDNA target and the length of the homologous RecA-ssDNA filament have important roles in the homology search. We discovered that as the end-to-end distance of the target dsDNA molecule is increased, constraining the available three-dimensional (3D) conformations of the molecule, the rate of homologous pairing decreases. Conversely, when the length of the ssDNA in the nucleoprotein filament is increased, homology is found faster. We propose a model for the DNA homology search process termed 'intersegmental contact sampling', in which the intrinsic multivalent nature of the RecA nucleoprotein filament is used to search DNA sequence space within 3D domains of DNA, exploiting multiple weak contacts to rapidly search for homology. Our findings highlight the importance of the 3D conformational dynamics of DNA, reveal a previously unknown facet of the homology search, and provide insight into the mechanism of DNA target location by this member of a universal family of proteins.
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Project description:The Bacillus subtilis recH342 strain, which decreases interspecies recombination without significantly affecting the frequency of transformation with homogamic DNA, carried a point mutation in the putative recX (yfhG) gene, and the mutation was renamed as recX342. We show that RecX (264 residues long), which shares partial identity with the Proteobacterial RecX (<180 residues), is a genuine recombination protein, and its primary function is to modulate the SOS response and to facilitate RecA-mediated recombinational repair and genetic recombination. RecX-YFP formed discrete foci on the nucleoid, which were coincident in time with RecF, in response to DNA damage, and on the poles and/or the nucleoid upon stochastic induction of programmed natural competence. When DNA was damaged, the RecX foci co-localized with RecA threads that persisted for a longer time in the recX context. The absence of RecX severely impaired natural transformation both with plasmid and chromosomal DNA. We show that RecX suppresses the negative effect exerted by RecA during plasmid transformation, prevents RecA mis-sensing of single-stranded DNA tracts, and modulates DNA strand exchange. RecX, by modulating the "length or packing" of a RecA filament, facilitates the initiation of recombination and increases recombination across species.
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Project description:In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions.
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Project description:Homologous recombination maintains genomic integrity by repairing broken chromosomes. The broken chromosome is partially resected to produce single-stranded DNA (ssDNA) that is used to search for homologous double-stranded DNA (dsDNA). This homology driven 'search and rescue' is catalyzed by a class of DNA strand exchange proteins that are defined in relation to Escherichia coli RecA, which forms a filament on ssDNA. Here, we review the regulation of RecA filament assembly and the mechanism by which RecA quickly and efficiently searches for and identifies a unique homologous sequence among a vast excess of heterologous DNA. Given that RecA is the prototypic DNA strand exchange protein, its behavior affords insight into the actions of eukaryotic RAD51 orthologs and their regulators, BRCA2 and other tumor suppressors.
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Project description:How does a folding protein negotiate a vast, featureless conformational landscape and adopt its native structure in biological real time? Motivated by this search problem, we developed a novel algorithm to compare protein structures. Procedures to identify structural analogs are typically conducted in three-dimensional space: the tertiary structure of a target protein is matched against each candidate in a database of structures, and goodness of fit is evaluated by a distance-based measure, such as the root-mean-square distance between target and candidate. This is an expensive approach because three-dimensional space is complex. Here, we transform the problem into a simpler one-dimensional procedure. Specifically, we identify and label the 11 most populated residue basins in a database of high-resolution protein structures. Using this 11-letter alphabet, any protein's three-dimensional structure can be transformed into a one-dimensional string by mapping each residue onto its corresponding basin. Similarity between the resultant basin strings can then be evaluated by conventional sequence-based comparison. The disorder ? order folding transition is abridged on both sides. At the onset, folding conditions necessitate formation of hydrogen-bonded scaffold elements on which proteins are assembled, severely restricting the magnitude of accessible conformational space. Near the end, chain topology is established prior to emergence of the close-packed native state. At this latter stage of folding, the chain remains molten, and residues populate natural basins that are approximated by the 11 basins derived here. In essence, our algorithm reduces the protein-folding search problem to mapping the amino acid sequence onto a restricted basin string.
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Project description:Mycobacteria have two genetically distinct pathways for the homology-directed repair of DNA double-strand breaks: homologous recombination (HR) and single-strand annealing (SSA). HR is abolished by deletion of RecA and reduced in the absence of the AdnAB helicase/nuclease. By contrast, SSA is RecA-independent and requires RecBCD. Here we examine the function of RecO in mycobacterial DNA recombination and repair. Loss of RecO elicits hypersensitivity to DNA damaging agents similar to that caused by deletion of RecA. We show that RecO participates in RecA-dependent HR in a pathway parallel to the AdnAB pathway. We also find that RecO plays a role in the RecA-independent SSA pathway. The mycobacterial RecO protein displays a zinc-dependent DNA binding activity in vitro and accelerates the annealing of SSB-coated single-stranded DNA. These findings establish a role for RecO in two pathways of mycobacterial DNA double-strand break repair and suggest an in vivo function for the DNA annealing activity of RecO proteins, thereby underscoring their similarity to eukaryal Rad52.
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Project description: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.
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