Language：English   Publishing type：Research paper (scientific journal)  

Homologous recombination (HR) is a universal mechanism operating in somatic and germ-line cells, where it contributes to the maintenance of genome stability and ensures the faithful distribution of genetic material, respectively. The ability to identify and exchange the strands of two homologous DNA molecules lies at the heart of HR and is mediated by RecA-family recombinases. Dmc1 is a meiosis-specific RecA homolog in eukaryotes, playing a predominant role in meiotic HR. However, Dmc1 cannot function without its two major auxiliary factor complexes, Swi5-Sfr1 and Hop2-Mnd1. Through biochemical reconstitutions, we demonstrate that Swi5-Sfr1 and Hop2-Mnd1 make unique contributions to stimulate Dmc1-driven strand exchange in a synergistic manner. Mechanistically, Swi5-Sfr1 promotes establishment of the Dmc1 nucleoprotein filament, whereas Hop2-Mnd1 defines a critical, rate-limiting step in initiating strand exchange. Following execution of this function, we propose that Swi5-Sfr1 then promotes strand exchange with Hop2-Mnd1. Thus, our findings elucidate distinct yet complementary roles of two auxiliary factors in Dmc1-driven strand exchange, providing mechanistic insights into some of the most critical steps in meiotic HR.

DOI： 10.1073/pnas.1917419117

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DOI： 10.1038/s41467-020-16750-3

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DOI： 10.1073/pnas.1917419117

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DOI： 10.7554/eLife.52566

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Publisher：Cold Spring Harbor Laboratory  

Abstract

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for second donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA family recombinases.

DOI： 10.1101/839324

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Hydrophobic base stacking is a major contributor to DNA double-helix stability. We report the discovery of specific unstacking effects in certain semihydrophobic environments. Water-miscible ethylene glycol ethers are found to modify structure, dynamics, and reactivity of DNA by mechanisms possibly related to a biologically relevant hydrophobic catalysis. Spectroscopic data and optical tweezers experiments show that base-stacking energies are reduced while base-pair hydrogen bonds are strengthened. We propose that a modulated chemical potential of water can promote "longitudinal breathing" and the formation of unstacked holes while base unpairing is suppressed. Flow linear dichroism in 20% diglyme indicates a 20 to 30% decrease in persistence length of DNA, supported by an increased flexibility in single-molecule nanochannel experiments in poly(ethylene glycol). A limited (3 to 6%) hyperchromicity but unaffected circular dichroism is consistent with transient unstacking events while maintaining an overall average B-DNA conformation. Further information about unstacking dynamics is obtained from the binding kinetics of large thread-intercalating ruthenium complexes, indicating that the hydrophobic effect provides a 10 to 100 times increased DNA unstacking frequency and an "open hole" population on the order of 10-2 compared to 10-4 in normal aqueous solution. Spontaneous DNA strand exchange catalyzed by poly(ethylene glycol) makes us propose that hydrophobic residues in the L2 loop of recombination enzymes RecA and Rad51 may assist gene recombination via modulation of water activity near the DNA helix by hydrophobic interactions, in the manner described here. We speculate that such hydrophobic interactions may have catalytic roles also in other biological contexts, such as in polymerases.

DOI： 10.1073/pnas.1909122116

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Publisher：Cold Spring Harbor Laboratory  

ABSTRACT

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. Here, we present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a widely conserved auxiliary factor. NMR and site-specific crosslinking experiments revealed two distinct sites within the intrinsically disordered N-terminus of Sfr1 that cooperatively bind to Rad51. Although disruption of this binding severely impaired Rad51 stimulation in vitro, interaction mutants did not show any defects in DNA repair. Unexpectedly, in the absence of the Rad51 paralogs Rad55-Rad57, which constitute another auxiliary factor complex, these interaction mutants were unable to promote DNA repair. Our findings provide molecular insights into Rad51 stimulation by Swi5-Sfr1 and suggest that, rather than functioning in an independent subpathway of HR as was previously proposed, Rad55-Rad57 facilitates the recruitment of Swi5-Sfr1 to Rad51.

DOI： 10.1101/738179

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DOI： 10.1073/pnas.1909122116

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DOI： 10.1017/S0033583518000094

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Mg2+ ion stimulates the DNA strand exchange reaction catalyzed by RecA, a key step in homologous recombination. To elucidate the molecular mechanisms underlying the role of Mg2+ and the strand exchange reaction itself, we investigated the interaction of RecA with Mg2+ and sought to determine which step of the reaction is affected. Thermal stability, intrinsic fluorescence, and native mass spectrometric analyses of RecA revealed that RecA binds at least two Mg2+ ions with KD ≈ 2 mM and 5 mM. Deletion of the C-terminal acidic tail of RecA made its thermal stability and fluorescence characteristics insensitive to Mg2+ and similar to those of full-length RecA in the presence of saturating Mg2+. These observations, together with the results of a molecular dynamics simulation, support the idea that the acidic tail hampers the strand exchange reaction by interacting with other parts of RecA, and that binding of Mg2+ to the tail prevents these interactions and releases RecA from inhibition. We observed that binding of the first Mg2+ stimulated joint molecule formation, whereas binding of the second stimulated progression of the reaction. Thus, RecA is actively involved in the strand exchange step as well as bringing the two DNAs close to each other.

DOI： 10.1093/nar/gky048

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DOI： 10.1093/nar/gky048

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DOI： 10.1038/s41594-017-0002-8

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DOI： 10.1038/s41594-017-0002-8

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DOI： 10.1017/S0033583517000099

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DOI： 10.1021/acs.biochem.5b00614

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DOI： 10.1093/nar/gkt1257

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DOI： 10.1093/nar/gks1131

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DOI： 10.1093/nar/gks140

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DOI： 10.1016/j.biochi.2010.08.006

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DOI： 10.1021/jm1002974

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DOI： 10.1073/pnas.0902234106

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DOI： 10.1073/pnas.0902723106

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DOI： 10.1002/cphc.200900253

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DOI： 10.1016/j.jmb.2008.08.030

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Tetracyclines coordinate metal(II) ions under physiological conditions forming chelate complexes with their ketoenolate moiety at rings B and C. These metal(II) complexes are the biologically relevant molecules conferring the antibiotic character of the drug by inhibiting ribosomal protein biosynthesis in prokaryotes. The Tet repressor, TetR, is the molecular switch for tetracycline resistance determinants in gram-negative bacteria. TetR controls transcription of a gene encoding the integral membrane protein TetA, which mediates active efflux of a tetracycline-metal(II) cation, [MeTc](+), by equimolar antiport with a proton. We evaluated distinct characteristics of the metal binding by crystal structure determination of TetR/[MeTc](+) complexes and of association equilibrium constants of [MeTc](+) and TetR/[MeTc](+) complexes. Various divalent metal ions bind to the same octahedral coordination site, defined by a histidine side chain of TetR, the tetracycline, and three water molecules. Whereas association constants for [MeTc](+) vary within 3 orders of magnitude, association of the [MeTc](+) cation to TetR is very similar for all measured divalent metals. Taking intracellular cation concentrations into account, it is evident that no other metal ion can compete with Mg(2+) for TetR/[MeTc](+) complex formation.

DOI： 10.1007/s00775-008-0395-2

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DOI： 10.1111/j.1365-2443.2008.01180.x

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DOI： 10.1093/nar/gkm752

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DOI： 10.1016/j.jmb.2006.10.042

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DOI： 10.1016/j.ab.2006.08.034

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DOI： 10.1016/j.gene.2006.05.020

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DOI： 10.1111/j.1742-4658.2006.05323.x

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DOI： 10.1053/j.gastro.2004.05.021

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Rad51 is a key element of recombinational DNA repair and its activity is regulated by phosphorylation of the tyrosine residue at position 315 by cAbl kinase. This phosphorylation could be involved in the resistance of cancer cells to chemotherapy. We have investigated the role of this residue by comparing the three-dimensional structures of human Rad51 and its prokaryotic homologue, Escherichia coli RecA. The residue appeared to be on the edge of the subunit-subunit interacting site. The fluorescence intensity of the tryptophan residue inserted at position 315 of human Rad51 in the place of tyrosine was decreased by adding 3 M urea, although the protein was not unfolded as there was no large change in the fluorescence peak position or circular dichroism signal. This change in fluorescence occurred at a lower urea concentration when the protein was diluted, which favours dissociation. These results indicate that the change is related to the dissociation of Rad51 polymer and that residue 315 is close to the subunit-subunit interacting site. ATP and ADP, which affect the filament structure, caused a blue shift in the fluorescence peak. These nucleotides probably altered the subunit-subunit contacts and may thus affect the filament structure. Phosphorylation of this residue could therefore affect the formation and structure of the Rad51 filament. Correct prediction of subunit-subunit interface of Rad51 by simple comparison of structures of Rad51 and RecA supports the idea that Rad51 forms the filament in a similar way as does RecA.

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Rad51, like its prokaryotic homolog RecA, forms a helical filament for homologous DNA recombination and recombinational DNA repair. Comparison of the three-dimensional structures of human Rad51 and Escherichia coli RecA indicated that the tyrosine residue at position 191 in human Rad51 lies at the centre of a putative subunit-subunit contact interface. We inserted a tryptophan residue as a fluorescent probe at the corresponding position in Xenopus Rad51.1 and found that its fluorescence depended upon the protein concentration, indicating that the residue is truly in the subunit-subunit interface. We also found that 3 M urea, which promoted the dissociation of Rad51 filament without complete unfolding of the protein, exposed the tryptophan residue to solvent. The fluorescence was not modified by binding to DNA and only slightly modified by ATP, indicating that the same site is used for formation of the active ATP-Rad51-DNA filament. The slight changes in fluorescence caused by ATP and ADP suggest that the subunit-subunit contact is altered, leading to the elongation of the filament by these nucleotides, as with the RecA filament. Thus, Rad51 forms filaments by subunit-subunit contact much like RecA does.

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Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.

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RecA protein requires ATP and its hydrolysis to ADP to complete the DNA strand-exchange reaction. We investigated how the nucleotides activate RecA by examining their effect on urea-induced unfolding, which could reflect domain-domain contact of protein. RecA is folded into three continuous domains: the N-terminal, central and C-terminal domains. The fluorescence of tyrosine residues, which lie mainly in the central domain, was modified in 1-3 M urea, while the red shift of fluorescence peak of the tryptophan residues located in the C-terminal domain occurred only in 3-6 M urea. Thus, the C-terminal domain of RecA is unfolded after the central part unfolds. The change in intensity of tryptophan fluorescence without a large shift in the peak at low concentrations of urea suggests that there are weak interactions between the central and C-terminal domains. This is supported by our observation that RecA protein lacking the C-terminal tail unfolded at lower concentrations of urea than the entire RecA, and with clear transitions, unlike the entire RecA. ATP and its unhydrolyzable analog (ATPgammaS), which enhance the binding of RecA to DNA, facilitated the urea-induced change in RecA tryptophan fluorescence, while ADP, an antagonist of ATP, prevented the change. ATP probably weakens the domain-domain contact and facilitates the DNA binding, while ADP stabilizes the contact and inhibits it. Supporting this conclusion, the binding of RecA lacking the C-terminal tail to DNA was not inhibited by ADP, while that of the intact RecA was.

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Human HsRad51 protein assembles on a DNA molecule through cooperative binding and forms a long filament for homologous recombination. We have characterized the self-assembly of HsRad51 by measuring the fluorescence resonance energy transfer from the fluorescein-labeled protein to the rhodamine-labeled protein. Self-assembly quickly reached equilibrium and can be described by the head-to-tail polymerization of monomers, like that of its procaryotic homologue, RecA. It depended strongly on pH and was inhibited by high salt concentrations, indicating that ionic interactions between negatively and positively charged aminoacid residues are important. By contrast, neither ATP nor ADP significantly affected the reaction.

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BACKGROUND: Human Rad51 protein (HsRad51) is a homologue of Escherichia coli RecA protein, and involved in homologous recombination. These eukaryotic and bacterial proteins catalyse strand exchange between two homologous DNA molecules, each forming a complex with single-stranded DNA (ssDNA) and ATP as the initial step. Both proteins hydrolyse ATP; however, the role of ATP hydrolysis appears to vary between the two proteins. RESULTS: Measurements using the fluorescence ssDNA analogue, poly(1,N6-etheno-deoxyadenosine), indicate that ATP affects the HsRad51-ssDNA complex, promoting two conformational states: one transient, rather rigid transition state and a final more flexible state. While ADP lowers the affinity of RecA protein to ssDNA, it is found to rather stabilize the HsRad51-ssDNA complex. ADP does not activate the strand exchange by HsRad51 but instead stimulates annealing between complementary ssDNAs. CONCLUSIONS: The hydrolysis of ATP promotes a transition of the HsRad51-ssDNA complex from a stiff state to less stiff state. The first state may be important for the strand separation of dsDNA in the initial step of strand exchange, while the second state may be important for annealing in the next step. However, hydrolysis does not dissociate HsRad51 from DNA as a component step of its recycling.

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Linear dichroism (LD) polarized-light spectroscopy is used to determine the arrangement of RecA in its large filamentous complex with DNA, active in homologous recombination. Angular orientation data for two tryptophan and seven tyrosine residues, deduced from differential LD of wild-type RecA vs. mutants that were engineered to attenuate the UV absorption of selected residues, revealed a rotation by some 40 degrees of the RecA subunits relative to the arrangement in crystal without DNA. In addition, conformational changes are observed for tyrosine residues assigned to be involved in DNA binding and in RecA-RecA contacts, thus potentially related to the global structure of the filament and its biological function. The presented spectroscopic approach, called "Site-Specific Linear Dichroism" (SSLD), may find forceful applications also to other biologically important fibrous complexes not amenable to x-ray crystallographic or NMR structural analysis.

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The structure of long filament of DNA-RecA protein complex, active in homologous recombination, has been assessed using linear dichroism (LD) spectroscopy. The angular orientation of the DNA nucleobases relative to the fibre axis was estimated to be 70 degrees from the unique LD band of etheno-modified poly(dA) at 320 nm. Angular orientation data for 2 tryptophan and 7 tyrosine residues of RecA were deduced from differential LD of wild type RecA versus modified proteins which were engineered to attenuate the UV absorption of selected residues. The results revealed a rotation by some 40 degrees of the RecA subunits relative to the arrangement in crystal without DNA. Conformational changes are also indicated for tyrosine residues assigned to be involved in DNA binding or in RecA-RecA contacts.

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DOI： 10.1006/jmbi.2000.4382

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DOI： 10.1006/jmbi.1998.2225

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DOI： 10.1111/j.1432-1033.1996.0091h.x

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