Structural and functional analyses of five conserved positively charged residues in the L1 and N-terminal DNA binding motifs of archaeal RADA protein

RecA family proteins engage in an ATP-dependent DNA strand exchange reaction that includes a ssDNA nucleoprotein helical filament and a homologous dsDNA sequence. In spite of more than 20 years of efforts, the molecular mechanism of homology pairing and strand exchange is still not fully understood. Here we report a crystal structure of Sulfolobus solfataricus RadA overwound right-handed filament with three monomers per helical pitch. This structure reveals conformational details of the first ssDNA binding disordered loop (denoted L1 motif) and the dsDNA binding N-terminal domain (NTD). L1 and NTD together form an outwardly open palm structure on the outer surface of the helical filament. Inside this palm structure, five conserved basic amino acid residues (K27, K60, R117, R223 and R229) surround a 25 A pocket that is wide enough to accommodate anionic ssDNA, dsDNA or both. Biochemical analyses demonstrate that these five positively charged residues are essential for DNA binding and for RadA-catalyzed D-loop formation. We suggest that the overwound right-handed RadA filament represents a functional conformation in the homology search and pairing reaction. A new structural model is proposed for the homologous interactions between a RadA-ssDNA nucleoprotein filament and its dsDNA target.     

Right or left turn? RecA family protein filaments promote homologous recombination through clockwise axial rotation

The RecA family proteins mediate homologous recombination, a ubiquitous mechanism for repairing DNA double-strand breaks (DSBs) and stalled replication forks. Members of this family include bacterial RecA, archaeal RadA and Rad51, and eukaryotic Rad51 and Dmc1. These proteins bind to single-stranded DNA at a DSB site to form a presynaptic nucleoprotein filament, align this presynaptic filament with homologous sequences in another double-stranded DNA segment, promote DNA strand exchange and then dissociate. It was generally accepted that RecA family proteins function throughout their catalytic cycles as right-handed helical filaments with six protomers per helical turn. However, we recently reported that archaeal RadA proteins can also form an extended right-handed filament with three monomers per helical turn and a left-handed protein filament with four monomers per helical turn. Subsequent structural and functional analyses suggest that RecA family protein filaments, similar to the F1-ATPase rotary motor, perform ATP-dependent clockwise axial rotation during their catalytic cycles. This new hypothesis has opened a new avenue for understanding the molecular mechanism of RecA family proteins in homologous recombination.     

Structure of the hDmc1-ssDNA Filament Reveals the Principles of Its Architecture

In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down''s, Klinefelter''s and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca²⁺, Mg²⁺, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination.     

Molecular visualization of the yeast Dmc1 protein ring and Dmc1-ssDNA nucleoprotein complex

Saccharomyces cerevisiae Dmc1, a meiosis-specific homologue of RecA, catalyzes homologous pairing and strand exchange during meiotic DNA recombination. The purified budding yeast Dmc1 (ScDmc1) protein exhibits much weaker recombinase activity in vitro as compared to that of the Escherichia coli RecA protein. Using atomic force microscopy (AFM) with carbon nanotube tips, we found ScDmc1 forms rings with an external diameter of 18 nm and a central cavity of 4 nm. In the presence of single-stranded DNA (ssDNA), the majority of the ScDmc1 protein (90%) bound DNA as protein rings; only a small faction (10%) was able to form filamentous structure. In contrast, nearly all RecA proteins form fine helical nucleoprotein filaments with ssDNA under identical conditions. RecA-mediated recombinase activity is initiated through the nucleation of RecA onto ssDNA to form helical nucleoprotein filaments. Our results support the notion that ScDmc1 becomes catalytically active only when it forms a helical nucleoprotein filament with ssDNA.     

Comparison of bacteriophage T4 UvsX and human Rad51 filaments suggests that RecA-like polymers may have evolved independently

The UvsX protein from bacteriophage T4 is a member of the RecA/Rad51/RadA family of recombinases active in homologous genetic recombination. Like RecA, Rad51 and RadA, UvsX forms helical filaments on DNA. We have used electron microscopy and a novel method for image analysis of helical filaments to show that UvsX-DNA filaments exist in two different conformations: an ADP state and an ATP state. As with RecA protein, these two states have a large difference in pitch. Remarkably, even though UvsX is only weakly homologous to RecA, both UvsX filament states are more similar to the RecA crystal structure than are RecA-DNA filaments. We use this similarity to fit the RecA crystal structure into the UvsX filament, and show that two of the three previously described blocks of similarity between UvsX and RecA are involved in the subunit-subunit interface in both the UvsX filament and the RecA crystal filament. Conversely, we show that human Rad51-DNA filaments have a different subunit-subunit interface than is present in the RecA crystal, and this interface involves two blocks of sequence similarity between Rad51 and RecA that do not overlap with those found between UvsX and RecA. This suggests that helical filaments in the RecA/Rad51/RadA family may have arisen from convergent evolution, with a conserved core structure that has assembled into multimeric filaments in a number of different ways.     

Structural and Functional Characterisation of a Conserved Archaeal RadA Paralog with Antirecombinase Activity

DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. RecA is the sole protein responsible for this reaction in bacteria, whereas there are several Rad51 paralogs that cooperate to catalyse strand exchange in eukaryotes. All archaea have at least one (and as many as four) RadA paralog, but their function remains unclear. Herein, we show that the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions and are not UV inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large archaeal RadC subfamily of archaeal RadA paralogs, functions as an ATPase that binds tightly to single-stranded DNA. However, Sso2452 is not an active recombinase in vitro and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.     

The Single-Stranded DNA Binding Protein of Sulfolobus solfataricus Acts in the Presynaptic Step of Homologous Recombination

Homologous recombination is an important pathway in the repair of DNA double-strand breaks in all organisms. In mesophiles, single-stranded DNA binding proteins (SSBs) are believed to be involved in the removal of single-stranded DNA (ssDNA) secondary structure during the presynaptic step of homologous recombination, facilitating the formation of a contiguous Rad51/RecA nucleoprotein filament. Here we report a role for the thermophilic archaeal Sulfolobus solfataricus SSB (SsoSSB) in the presynaptic step of homologous recombination. We have identified multiple quaternary structural forms of this protein in vivo and examined the activity of SsoSSB with the strand-exchange protein S. solfataricus RadA (SsoRadA). Using gel-shift analysis, we found that the two major forms of SsoSSB have different DNA binding affinities and site sizes. Biochemical examination of the monomeric form of SsoSSB suggests that it has a minor role in presynapsis and may slightly inhibit the ssDNA-dependent ATPase activity of SsoRadA. The tetrameric form of SsoSSB, however, significantly inhibits SsoRadA ssDNA-dependent ATPase activity under both saturating and subsaturating conditions. Order-of-addition experiments indicate that preincubation of tetrameric SsoSSB and SsoRadA prior to reaction initiation with ssDNA relieves the inhibition observed when SsoSSB is added either before or after SsoRadA. In addition, we demonstrate a direct interaction between SsoRadA and SsoSSB using coimmunoprecipitation. Taken together, these results suggest that a direct interaction between SsoSSB and SsoRadA may occur in vivo prior to the formation of the SsoRadA nucleoprotein filament.     

Elastic behavior of RecA-DNA helical filaments

Escherichia coli RecA protein forms a right-handed helical filament with DNA molecules and has an ATP-dependent activity that exchanges homologous strands between single-stranded DNA (ssDNA) and duplex DNA. We show that the RecA-ssDNA filamentous complex is an elastic helical molecule whose length is controlled by the binding and release of nucleotide cofactors. RecA-ssDNA filaments were fluorescently labelled and attached to a glass surface inside a flow chamber. When the chamber solution was replaced by a buffer solution without nucleotide cofactors, the RecA-ssDNA filament rapidly contracted approximately 0.68-fold with partial filament dissociation. The contracted filament elongated up to 1.25-fold when a buffer solution containing ATPgammaS was injected, and elongated up to 1.17-fold when a buffer solution containing ATP or dATP was injected. This contraction-elongation behavior was able to be repeated by the successive injection of dATP and non-nucleotide buffers. We propose that this elastic motion couples to the elastic motion and/or the twisting rotation of DNA strands within the filament by adjusting their helical phases.     

Does a stretched DNA structure dictate the helical geometry of RecA-like filaments?

Proteins in the RecA/Rad51/RadA/UvsX family form helical filaments on DNA in which the DNA is stretched and untwisted. A comparison of the average helical parameters of these filaments from five different proteins, obtained from archaea, eubacteria and eukaryotes, suggests that an intrinsic state of DNA may be responsible for the conservation of these particular filament forms across evolution. In this view, these proteins stabilize this existing state of DNA, rather than induce a novel conformation.     

Dynamics of RecA filaments on single-stranded DNA

RecA, the key protein in homologous recombination, performs its actions as a helical filament on single-stranded DNA (ssDNA). ATP hydrolysis makes the RecA–ssDNA filament dynamic and is essential for successful recombination. RecA has been studied extensively by single-molecule techniques on double-stranded DNA (dsDNA). Here we directly probe the structure and kinetics of RecA interaction with its biologically most relevant substrate, long ssDNA molecules. We find that RecA ATPase activity is required for the formation of long continuous filaments on ssDNA. These filaments both nucleate and extend with a multimeric unit as indicated by the Hill coefficient of 5.4 for filament nucleation. Disassembly rates of RecA from ssDNA decrease with applied stretching force, corresponding to a mechanism where protein-induced stretching of the ssDNA aids in the disassembly. Finally, we show that RecA–ssDNA filaments can reversibly interconvert between an extended, ATP-bound, and a compressed, ADP-bound state. Taken together, our results demonstrate that ATP hydrolysis has a major influence on the structure and state of RecA filaments on ssDNA.     

Conformational flexibility revealed by the crystal structure of a crenarchaeal RadA

Homologous recombinational repair is an essential mechanism for repair of double-strand breaks in DNA. Recombinases of the RecA-fold family play a crucial role in this process, forming filaments that utilize ATP to mediate their interactions with single- and double-stranded DNA. The recombinase molecules present in the archaea (RadA) and eukaryota (Rad51) are more closely related to each other than to their bacterial counterpart (RecA) and, as a result, RadA makes a suitable model for the eukaryotic system. The crystal structure of Sulfolobus solfataricus RadA has been solved to a resolution of 3.2 Å in the absence of nucleotide analogues or DNA, revealing a narrow filamentous assembly with three molecules per helical turn. As observed in other RecA-family recombinases, each RadA molecule in the filament is linked to its neighbour via interactions of a short β-strand with the neighbouring ATPase domain. However, despite apparent flexibility between domains, comparison with other structures indicates conservation of a number of key interactions that introduce rigidity to the system, allowing allosteric control of the filament by interaction with ATP. Additional analysis reveals that the interaction specificity of the five human Rad51 paralogues can be predicted using a simple model based on the RadA structure.     

Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins

The RecA family of proteins mediates homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against DNA double strand breaks. RecA proteins are thought to facilitate DNA strand exchange reactions as closed-rings or as right-handed helical filaments. Here, we report the crystal structure of a left-handed Sulfolobus solfataricus RadA helical filament. Each protomer in this left-handed filament is linked to its neighbour via interactions of a β-strand polymerization motif with the neighbouring ATPase domain. Immediately following the polymerization motif, we identified an evolutionarily conserved hinge region (a subunit rotation motif) in which a 360° clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the AMP–PNP right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. Additional structural and functional analyses of wild-type and mutant proteins confirmed that the subunit rotation motif is crucial for enzymatic functions of RecA family proteins. These observations support the hypothesis that RecA family protein filaments may function as rotary motors.     

The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange, it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that single-stranded DNA-binding protein can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site.     

Analytical model for determination of parameters of helical structures in solution by small angle scattering: comparison of RecA structures by SANS

The filament structures of the self-polymers of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, their complexes with ATPgammaS, phage M13 single-stranded DNA (ssDNA) and the tertiary complexes RecA::ATPgammaS::ssDNA were compared by small angle neutron scattering. A model was developed that allowed for an analytical solution for small angle scattering on a long helical filament, making it possible to obtain the helical pitch and the mean diameter of the protein filament from the scattering curves. The results suggest that the structure of the filaments formed by these two RecA proteins, and particularly their complexes with ATPgammaS, is conservative.     

Conserved conformation of RecA protein after executing the DNA strand-exchange reaction. A site-specific linear dichroism structure study

RecA protein and its eukaryotic homologue Rad51 protein catalyzes the DNA strand exchange, which is a key reaction of homologous recombination. At the initial step of the reaction, RecA proteins form a helical filament on a single-stranded DNA (ssDNA). Binding of double-stranded DNA (dsDNA) to the filament triggers the homology search; as homology is found, the exchange of strands occurs, and the displaced DNA is released. These are the principal steps of genetic recombination; however, despite many years of extensive study of RecA activities, the details of the mechanism are still obscure. A high-resolution structure of the active nucleoprotein filament could provide information to help understand this process. Using a linear dichroism polarized-light spectroscopy technique, in combination with protein engineering (the site-specific linear dichroism method), we have previously studied the arrangement of RecA in complex with ssDNA. In the present study, we have used this approach to search for structural variations of RecA at the atomic level as the DNA in the complex is changed from ssDNA to dsDNA. The structural data of the RecA-dsDNA filament are found to be very similar to the data previously obtained for the RecA-ssDNA complex, indicating that the overall orientation and also the internal structure of RecA in the active filament are not markedly altered when the bound DNA changes from single- to double-stranded. The implications of the structural similarities as well as the significance of some conformational variations observed for a few amino acid residues that may be involved in interactions with DNA are discussed.     

The mutant RecA proteins, RecAR243Q and RecAK245N, exhibit defective DNA binding in homologous pairing

In homologous pairing, the RecA protein sequentially binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), aligning the two DNA molecules within the helical nucleoprotein filament. To identify the DNA binding region, which stretches from the outside to the inside of the filament, we constructed two mutant RecA proteins, RecAR243Q and RecAK245N, with the amino acid substitutions of Arg243 to Gln and Lys245 to Asn, respectively. These amino acids are exposed to the solvent in the crystal structure of the RecA protein and are located in the central domain, which is believed to be the catalytic center of the homologous pairing activity. The mutations of Arg243 to Gln (RecAR243Q) and Lys245 to Asn (RecAK245N) impair the repair of UV-damaged DNA in vivo and cause defective homologous pairing of ssDNA and dsDNA in vitro. Although RecAR243Q is only slightly defective and RecAK245N is completely proficient in ssDNA binding to form the presynaptic filament, both mutant RecA proteins are defective in the formation of the three-component complex including ssDNA, dsDNA, and RecA protein. The ability to form dsDNA from complementary single strands is also defective in both RecAR243Q and RecAK245N. These results suggest that the region including Arg243 and Lys245 may be involved in the path of secondary DNA binding to the presynaptic filament.     

Molecular anatomy of the recombination mediator function of Saccharomyces cerevisiae Rad52

A helical filament of Rad51 on single-strand DNA (ssDNA), called the presynaptic filament, catalyzes DNA joint formation during homologous recombination. Rad52 facilitates presynaptic filament assembly, and this recombination mediator activity is thought to rely on the interactions of Rad52 with Rad51, the ssDNA-binding protein RPA, and ssDNA. The N-terminal region of Rad52, which has DNA binding activity and an oligomeric structure, is thought to be crucial for mediator activity and recombination. Unexpectedly, we find that the C-terminal region of Rad52 also harbors a DNA binding function. Importantly, the Rad52 C-terminal portion alone can promote Rad51 presynaptic filament assembly. The middle portion of Rad52 associates with DNA-bound RPA and contributes to the recombination mediator activity. Accordingly, expression of a protein species that harbors the middle and C-terminal regions of Rad52 in the rad52 Delta327 background enhances the association of Rad51 protein with a HO-made DNA double-strand break and partially complements the methylmethane sulfonate sensitivity of the mutant cells. Our results provide a mechanistic framework for rationalizing the multi-faceted role of Rad52 in recombination and DNA repair.     

The stretched DNA geometry of recombination and repair nucleoprotein filaments

The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA x tsDNA complex), has been elucidated by classical methods. Here we review the systematic characterization of the helical geometries of the three DNA strands of the RecA x tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. Measurements of the helical parameters for the RecA x tsDNA complex are consistent with the hypothesis that this complex is a late, poststrand-exchange intermediate with the outgoing strand shifted by about three base pairs with respect to its registry with the incoming and complementary strands. All three strands in the RecA x tsDNA complex adopt extended and unwound conformations similar to those of RecA-bound ssDNA and dsDNA.     

The Rad51/RadA N-terminal domain activates nucleoprotein filament ATPase activity

Proteins in the RecA/RadA/Rad51 family form helical filaments on DNA that function in homologous recombination. While these proteins all have the same highly conserved ATP binding core, the RadA/Rad51 proteins have an N-terminal domain that shows no homology with the C-terminal domain found in RecA. Both the Rad51 N-terminal and RecA C-terminal domains have been shown to bind DNA, but no role for these domains has been established. We show that RadA filaments can be trapped in either an inactive or active conformation with respect to the ATPase and that activation involves a large rotation of the subunit aided by the N-terminal domain. The G103E mutation within the yeast Rad51 N-terminal domain inactivates the filament by failing to make proper contacts between the N-terminal domain and the core. These results show that the N-terminal domains play a regulatory role in filament activation and highlight the modular architecture of the recombination proteins.     

Insights into the mechanism of Rad51 recombinase from the structure and properties of a filament interface mutant

Rad51 protein promotes homologous recombination in eukaryotes. Recombination activities are activated by Rad51 filament assembly on ssDNA. Previous studies of yeast Rad51 showed that His352 occupies an important position at the filament interface, where it could relay signals between subunits and active sites. To investigate, we characterized yeast Rad51 H352A and H352Y mutants, and solved the structure of H352Y. H352A forms catalytically competent but salt-labile complexes on ssDNA. In contrast, H352Y forms salt-resistant complexes on ssDNA, but is defective in nucleotide exchange, RPA displacement and strand exchange with full-length DNA substrates. The 2.5 Å crystal structure of H352Y reveals a right-handed helical filament in a high-pitch (130 Å) conformation with P6₁ symmetry. The catalytic core and dimer interface regions of H352Y closely resemble those of DNA-bound Escherichia coli RecA protein. The H352Y mutation stabilizes Phe187 from the adjacent subunit in a position that interferes with the γ-phosphate-binding site of the Walker A motif/P-loop, potentially explaining the limited catalysis observed. Comparison of Rad51 H352Y, RecA–DNA and related structures reveals that the presence of bound DNA correlates with the isomerization of a conserved cis peptide near Walker B to the trans configuration, which appears to prime the catalytic glutamate residue for ATP hydrolysis.