Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules.
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ABSTRACT:
SUBMITTER: Hilario J
PROVIDER: S-EPMC2613362 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The human DNA repair protein RAD51 is the crucial component of helical nucleoprotein filaments that drive homologous recombination. The molecular mechanistic details of how this structure facilitates the requisite DNA strand rearrangements are not known but must involve dynamic interactions between RAD51 and DNA. Here, we report the real-time kinetics of human RAD51 filament assembly and disassembly on individual molecules of both single- and double-stranded DNA, as measured using magnetic tweezers. The relative rates of nucleation and filament extension are such that the observed filament formation consists of multiple nucleation events that are in competition with each other. For varying concentration of RAD51, a Hill coefficient of 4.3 +/- 0.5 is obtained for both nucleation and filament extension, indicating binding to dsDNA with a binding unit consisting of multiple (> or =4) RAD51 monomers. We report Monte Carlo simulations that fit the (dis)assembly data very well. The results show that, surprisingly, human RAD51 does not form long continuous filaments on DNA. Instead each nucleoprotein filament consists of a string of many small filament patches that are only a few tens of monomers long. The high flexibility and dynamic nature of this arrangement is likely to facilitate strand exchange.
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Project description:Rad51 is a core component of the eukaryotic homologous recombination machinery and is responsible for key mechanistic steps during strand invasion. Higher order oligomers of Rad51 display a remarkable degree of structural variation, forming rings, compressed filaments, and elongated filaments. It is unclear whether Rad51 can transition directly between these different oligomeric structures without disassembling first into monomers. We have used single-molecule microscopy to investigate the behavior of human Rad51 assembled on double-stranded DNA. Our results show that human Rad51 can form elongated nucleoprotein filaments on DNA, but ATP hydrolysis causes a decrease in their length without concomitant dissociation of protein. Compressed Rad51 filaments can re-elongate when presented with either ATP or the non-hydrolyzable analog AMP-PNP, and these cycles of elongation and compression are reversible. A Rad51 mutant deficient in ATP hydrolysis is locked into an extended conformation that is incapable of transitioning to a compressed filament. Similarly, wild-type Rad51 bound to DNA in the presence of AMP-PNP was trapped in the elongated state. Proteins incapable of transitioning to the compressed state were also highly resistant to dissociation from the DNA. Taken together, our results indicate that nucleotide hydrolysis by human Rad51 triggers a reversible structural transition leading to filaments with reduced helical pitch.
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Project description:Homologous recombination is a conserved pathway for repairing double-stranded breaks, which are processed to yield single-stranded DNA overhangs that serve as platforms for presynaptic-complex assembly. Here we use single-molecule imaging to reveal the interplay between Saccharomyces cerevisiae RPA, Rad52 and Rad51 during presynaptic-complex assembly. We show that Rad52 binds RPA-ssDNA and suppresses RPA turnover, highlighting an unanticipated regulatory influence on protein dynamics. Rad51 binding extends the ssDNA, and Rad52-RPA clusters remain interspersed along the presynaptic complex. These clusters promote additional binding of RPA and Rad52. Our work illustrates the spatial and temporal progression of the association of RPA and Rad52 with the presynaptic complex and reveals a new RPA-Rad52-Rad51-ssDNA intermediate, with implications for how the activities of Rad52 and RPA are coordinated with Rad51 during the later stages of recombination.
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Project description:Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.
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Project description:The unique translocation and DNA unwinding properties of DNA helicases can be concealed by the stochastic behavior of enzyme molecules within the necessarily large populations used in ensemble experiments. With recent technological advances, the direct visualization of helicases acting on individual DNA molecules has contributed significantly to the current understanding of their mechanisms of action and biological functions. The combination of single-molecule techniques that enable both manipulation of individual protein or DNA molecules and visualization of their actions has made it possible to literally see novel and unique biochemical characteristics that were previously masked. Here, we describe the execution and use of single-molecule fluorescence imaging techniques, focusing on methods that include optical trapping in conjunction with epifluorescent imaging, and also surface immobilization in conjunction with total internal reflection fluorescence visualization. Combined with microchannel flow cells and microfluidic control, these methods allow individual fluorescently labeled protein and DNA molecules to be imaged and tracked, affording measurement of DNA unwinding and translocation at single-molecule resolution.
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Project description:Formation of RAD51 filaments on single-stranded DNA is an essential event during homologous recombination, which is required for homology search, strand exchange and protection of replication forks. Formation of nucleoprotein filaments (NF) is required for development and genomic stability, and its failure is associated with developmental abnormalities and tumorigenesis. Here we describe the structure of the human RAD51 NFs and of its Walker box mutants using electron microscopy. Wild-type RAD51 filaments adopt an 'open' conformation when compared to a 'closed' structure formed by mutants, reflecting alterations in helical pitch. The kinetics of formation/disassembly of RAD51 filaments show rapid and high ssDNA coverage via low cooperativity binding of RAD51 units along the DNA. Subsequently, a series of isomerization or dissociation events mediated by nucleotide binding state creates intrinsically dynamic RAD51 NFs. Our findings highlight important a mechanistic divergence among recombinases from different organisms, in line with the diversity of biological mechanisms of HR initiation and quality control. These data reveal unexpected intrinsic dynamic properties of the RAD51 filament during assembly/disassembly, which may be important for the proper control of homologous recombination.
| S-EPMC5934667 | biostudies-literature
Project description:Human Rad51 (hRad51), the protein central to DNA pairing and strand exchange during homologous recombination, polymerizes on DNA to form nucleoprotein filaments. By making use of magnetic tweezers to manipulate individual DNA molecules, we measured the nucleation and growth of hRad51 nucleoprotein filaments, and their subsequent disassembly in real time. The dependence of the initial polymerization rate upon the concentration of hRad51 suggests that the rate-limiting step is the formation of a nucleus involving 5.5 +/- 1.5 hRad51 monomers, corresponding to one helical turn of the hRad51 nucleoprotein filament. Polymerization is highly cooperative (i.e. a nucleation-limited reaction) at low concentrations and less cooperative (a growth-limited reaction) at high concentrations of the protein. We show that the observed preference of hRad51 to form nucleoprotein filaments on double-stranded DNA rather than on single-stranded DNA is due to the fact that it depolymerizes much faster from ssDNA than from dsDNA: indeed, hRad51 polymerizes faster on ssDNA than on dsDNA. Hydrolysis of ATP by hRad51 does not correlate with its dissociation from dsDNA. This suggests that hRad51 does not depolymerize rapidly from dsDNA after strand exchange but stays bound to the heteroduplex, highlighting the importance of partner proteins to facilitate hRad51 depolymerization from dsDNA.
| S-EPMC2175369 | biostudies-literature
Project description:RAD51 mediates homologous recombination by forming an active DNA nucleoprotein filament (NPF). A conserved aspartate that forms a salt bridge with the ATP ?-phosphate is found at the nucleotide-binding interface between RAD51 subunits of the NPF known as the ATP cap. The salt bridge accounts for the nonphysiological cation(s) required to fully activate the RAD51 NPF. In contrast, RecA homologs and most RAD51 paralogs contain a conserved lysine at the analogous structural position. We demonstrate that substitution of human RAD51(Asp-316) with lysine (HsRAD51(D316K)) decreases NPF turnover and facilitates considerably improved recombinase functions. Structural analysis shows that archaebacterial Methanococcus voltae RadA(D302K) (MvRAD51(D302K)) and HsRAD51(D316K) form extended active NPFs without salt. These studies suggest that the HsRAD51(Asp-316) salt bridge may function as a conformational sensor that enhances turnover at the expense of recombinase activity.
| S-EPMC3308741 | biostudies-literature
Project description:The human recombinase hRad51 is a key protein for the maintenance of genome integrity and for cancer development. Polymerization and depolymerization of hRad51 on duplex DNA were studied here using a new generation of magnetic tweezers, measuring DNA twist in real time with a resolution of 5 degrees . Our results combined with earlier structural information suggest that DNA is somewhat less extended by hRad51 than by RecA (4.5 vs. 5.1 A per base pair) and untwisted by 18.2 degrees per base pair. They also confirm a stoichiometry of 3-4 bp per protein in the hRad51-dsDNA nucleoprotein filament. At odds with earlier claims, we show that after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition/release of single proteins, involving DNA twisting steps of 65 degrees +/- 5 degrees. Simple numeric simulations show that this mechanism is an efficient way to minimize nucleoprotein filament defects. Nucleoprotein filament growth from a preformed nucleus was observed at hRad51 concentrations down to 10 nM, whereas nucleation was never observed below 100 nM in the same buffer. This behavior can be associated with the different stoichiometries of nucleation and growth. It may be instrumental in vivo to permit efficient continuation of strand exchange by hRad51 alone while requiring additional proteins such as Rad52 for its initiation, thus keeping the latter under the strict control of regulatory pathways.
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Project description:The central catalyst in eukaryotic ATP-dependent homologous recombination consists of RAD51 proteins, polymerized around single-stranded DNA. This nucleoprotein filament recognizes and invades a homologous duplex DNA segment. After strand exchange, the nucleoprotein filament should disassemble so that the recombination process can be completed. The molecular mechanism of RAD51 filament disassembly is poorly understood. Here we show, by combining optical tweezers with single-molecule fluorescence microscopy and microfluidics, that disassembly of human RAD51 nucleoprotein filaments results from the interplay between ATP hydrolysis and the release of the tension stored in the filament. By applying external tension to the DNA, we found that disassembly slows down and can even be stalled. We quantified the fluorescence of RAD51 patches and found that disassembly occurs in bursts interspersed by long pauses. After relaxation of a stalled complex, pauses were suppressed resulting in a large burst. These results indicate that tension-dependent disassembly takes place only from filament ends, after tension-independent ATP hydrolysis. This integrative single-molecule approach allowed us to dissect the mechanism of this principal homologous recombination reaction step, which in turn clarifies how disassembly can be influenced by accessory proteins.
| S-EPMC3871861 | biostudies-literature