Mechanism Of Strand Exchange Research Articles

The archaic chaperone-usher pathways may depend on donor strand exchange for intersubunit interactions.

Subunit-subunit interactions of the classical and alternate chaperone-usher (CU) systems have been shown to proceed through a donor strand exchange (DSE) mechanism. However, it is not known whether DSE is required for intersubunit interactions in the archaic CU system. We have previously shown that the Myxococcus xanthus Mcu system, a member of the archaic CU family that functions in spore coat formation, is likely to use the principle of donor strand complementation to medicate chaperone-subunit interactions analogous to the classical CU pathway. Here we describe the results of studies on Mcu subunit-subunit interactions. We constructed a series of N-terminal-deleted, single amino acid-mutated and donor strand-complemented Mcu subunits, and characterized their abilities to participate in subunit-subunit interactions. It appears that certain residues in both the N and C termini of McuA, a subunit of the Mcu system, play a critical role in intersubunit interactions and these interactions may involve the general principle of DSE of the classical and alternate CU systems. In addition, the specificity of the M. xanthus CU system for Mcu subunits over other spore coat proteins is demonstrated.

PH-dependent Activities and Structural Stability of Loop-2-anchoring Helix of RadA Recombinase from Methanococcus voltae

RadA is an archaeal orthologue of human recombinase Rad51. This superfamily of recombinases, which also includes eukaryal meiosis-specific DMC1 and remotely related bacterial RecA, form filaments on single-stranded DNA in the presence of ATP and promote a strand exchange reaction between the single-stranded DNA and a homologous double-stranded DNA. Due to its feasibility of getting crystals and similarity (> 40% sequence identity) to eukaryal homologues, we have studied RadA from Methanococcus voltae (MvRadA) as a structural model for understanding the molecular mechanism of homologous strand exchange. Here we show this protein’s ATPase and strand exchange activities are minimal at pH 6.0. Interestingly, MvRadA’s pH dependence is similar to the properties of human Rad51 but dissimilar to that of the well-studied E. coli RecA. A structure subsequently determined at pH 6.0 reveals features indicative of an ATPase-inactive form with a disordered L2 loop. Comparison with a previously determined ATPase-active form at pH 7.5 implies that the stability of the ATPase-active conformation is reduced at the acidic pH. We interpret these results as further suggesting an ordered disposition of the DNA-binding L2 region, similar to what has been observed in the previously observed ATPase-active conformation, is required for promoting hydrolysis of ATP and strand exchange between single- and double-stranded DNA. His-276 in the mobile L2 region was observed to be partially responsible for the pH-dependent activities of MvRadA.

Organization of DNA Partners and Strand Exchange Mechanisms during Flp Site-Specific Recombination Analyzed by Difference Topology, Single Molecule FRET and Single Molecule TPM

Flp site-specific recombination between two target sites (FRTs) harboring non-homology within the strand exchange region does not yield stable recombinant products. In negatively supercoiled plasmids containing head-to-tail sites, the reaction produces a series of knots with odd-numbered crossings. When the sites are in head-to-head orientation, the knot products contain even-numbered crossings. Both types of knots retain parental DNA configuration. By carrying out Flp recombination after first assembling the topologically well defined Tn3 resolvase synapse, it is possible to determine whether these knots arise by a processive or a dissociative mechanism. The nearly exclusive products from head-to-head and head-to-tail oriented “non-homologous” FRT partners are a 4-noded knot and a 5-noded knot, respectively. The corresponding products from a pair of native (homologous) FRT sites are a 3-noded knot and a 4-noded catenane, respectively. These results are consistent with non-homology-induced two rounds of dissociative recombination by Flp, the first to generate reciprocal recombinants containing non-complementary base pairs and the second to produce parental molecules with restored base pairing. Single molecule fluorescence resonance energy transfer (smFRET) analysis of geometrically restricted FRTs, together with single molecule tethered particle motion (smTPM) assays of unconstrained FRTs, suggests that the sites are preferentially synapsed in an anti-parallel fashion. This selectivity in synapse geometry occurs prior to the chemical steps of recombination, signifying early commitment to a productive reaction path. The cumulative topological, smFRET and smTPM results have implications for the relative orientation of DNA partners and the directionality of strand exchange during recombination mediated by tyrosine site-specific recombinases.

Observation of Synapse Rotation of Engineered Recombinase HIN-H107Y

Site-specific recombination promotes integration, excision or inversion of DNA strands during gene transcription and chromosome replication. The recombination process generally involves the formation of synaptic complexes, DNA cleavage and rejoining, though the specific mechanism of each enzyme varies. The Hin recombinase is a member of the serine recombinase family which has an active serine residue that can catalyze the DNA cleavage and re-ligating. Hin functions to invert a 900 bp DNA segment between two hin-binding (hix) sites within the Salmonella chromosome that contains a promoter for downstream flagellar genes. The inversion requires the auxiliary protein Fis and two Fis-binding sites on the targeted DNA segment, which assemble together with hix sites and Hin to form an invertasome structure. The assembly is stimulated by the protein HU. A class of engineered Hin mutants Hin-H107Y (H107Y) is able to assemble oligonucleotide substrates containing hix sites into stable synaptic complexes that catalyze recombination without Fis and HU. Structural data suggest that Hin share with gamma-delta resolvase a similar helix-turn-helix domain at the 40-50 amino acid residues. We hypothesized that H107Y could act like resolvase to form four subunits synapse, cleave both strands of DNA within the center of hix site, rotate and rejoin. We therefore used single DNA assays to analyze the dynamics associated with H107Y catalyzed strand exchange.Enzyme-DNA binding and synapse formation was observed with a single DNA containing two hix sites. We further obtained data for subunits rotation and re-ligation from a braiding assay which consisted of double DNAs each having a single hix site, indicating that the rotation strand exchange mechanism observed recently for Bxb1 Int is also possessed by Hin.

Sequence-specific synthesis of macromolecules using DNA-templated chemistry

Using a strand exchange mechanism we have prepared, by DNA templated chemistry, two 10-mers with defined and tunable monomer sequences. An optimized reaction protocol achieves 85% coupling yield per step, demonstrating that DNA-templated chemistry is a powerful tool for the synthesis of macromolecules with full sequence control.

The Structure of a Monomeric Mutant Cks Protein Reveals Multiple Functions for a Conserved Hinge-Region Proline

Cks (cyclin-dependent kinase subunit) proteins are essential eukaryotic cell cycle regulatory proteins that physically associate with cyclin-dependent kinases (Cdks) to modulate their activity. Cks proteins have also been studied for their ability to form domain-swapped dimers by exchanging β-strands. Domain swapping is mediated by a conserved β-hinge region containing two proline residues. Previous structural studies indicate that Cks in its dimer form is unable to bind Cdk, suggesting that the monomer–dimer equilibrium of Cks may have an effect on Cks-mediated Cdk regulation. We present the crystal structure of a proline-to-alanine mutant Saccharomyces cerevisiae Cks protein (Cks1 P93A) that preferentially adopts the monomer conformation but surprisingly fails to bind Cdk. Comparison of the Cks1 P93A structure to that of other Cks proteins reveals that Pro93 is critical for stabilizing a multiple β-turn structure in the hinge region that properly positions an essential Cdk-binding residue. Additionally, we find that these β-turn formations, conserved in Cks homologs, have implications for the mechanism and preferentiality of strand exchange. Together, our observations suggest that the conservation of Cks hinge-region prolines reflects their functions in forming a Cdk binding interface and that the ability of these prolines to control partitioning between monomer and dimer is a consequence of the β-turn networks within the hinge.

Postreplication repair mechanisms in the presence of DNA adducts in Escherichia coli

During bacterial replication, DNA polymerases may encounter DNA lesions that block processive DNA synthesis. Uncoupling the replicative helicase from the stalled DNA polymerase results in the formation of single-stranded DNA (ssDNA) gaps, which are repaired by postreplication repair (PRR), a process that involves at least three mechanisms that collectively remove, circumvent or bypass lesions. RecA mediated excision repair (RAMER) and homologous recombination (HR) are strand-exchange mechanisms that appear to be the predominant strategies for gap repair in the absence of prolonged SOS induction. During RAMER, RecA mediates pairing of damaged ssDNA with an undamaged homologous duplex and subsequent exchange of strands between the damaged and undamaged DNA. Repair of the lesion occurs in the context of the strand-exchange product and is initiated by UvrABC excinuclease; the resulting patch is filled by DNA synthesis using the complementary strand of the homologous duplex as a template. HR uses a complementary strand of an undamaged homologous duplex as a transient template for DNA synthesis. HR requires the formation and resolution of Holliday junctions, and is a mechanism to circumvent the lesion; lesions persisting in one of the daughter DNA duplexes will normally be repaired prior to subsequent rounds of replication/cell division. Translesion DNA Synthesis (TLS) does not involve strand-exchange mechanisms; it is carried out by specialized DNA polymerases that are able to catalyze nucleotide incorporation opposite lesions that cannot be bypassed by high-fidelity replicative polymerases. Maximum levels of TLS occur during prolonged SOS induction generally associated with increased mutagenesis. RAMER, HR and TLS are alternative mechanisms for processing a common intermediate-the ssDNA gap containing a RecA nucleofilament. The actual pathway that is utilized will be strongly influenced by multiple factors, including the blocking/coding capacity of the lesion, the nature of the gene products that can be assembled at the ssDNA gap, the availability of a homologous partner for RAMER and HR, and protein:protein interactions and post-translational modifications that modulate the mutagenic activity of Pol-IV and Pol-V.

Single-molecule analysis reveals the molecular bearing mechanism of DNA strand exchange by a serine recombinase

Structural and topological data suggest that serine site-specific DNA recombinases exchange duplex DNAs by rigid-body relative rotation of the two halves of the synapse, mediated by a flat protein-protein interaction surface. We present evidence for this rotational motion for a simple serine recombinase, the Bxb1 phage integrase, from a single-DNA-based supercoil-release assay that allows us to follow crossover site cleavage, rotation, religation, and product release in real time. We have also used a two-DNA braiding-relaxation experiment to observe the effect of synapse rotation in reactions on two long molecules. Relaxation and unbraiding are rapid (averaging 54 and 70 turns/s, respectively) and complete, with no discernible pauses. Nevertheless, the molecular friction associated with rotation is larger than that of type-I topoisomerases in a similar assay. Surprisingly we find that the synapse can stay rotationally "open" for many minutes.

Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

Homologous recombination (HR), a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR) processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR). T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms.

Nuclear localization and antisense effect of PNA internalized by ASGP-R-mediated endocytosis with protein/DNA conjugates

In order for peptide nucleic acids (PNAs) to be effective as therapeutic agents, methods for cellular delivery must be developed. Here we demonstrate spontaneous nuclear localization and antisense effects of peptide nucleic acids (PNAs) delivered to hepatic cells through asialoglycoprotein receptor-mediated endocytosis. Asialofetuin conjugates with DNA oligonucleotides (AF/DNA) complementary to the PNA of interest were designed as cell-specific delivery vectors. PNAs hybridized to the asialofetuin–oligonucleotide conjugates were internalized into murine primary hepatocytes and human HepG2 hepatocarcinoma cells effectively through receptor-mediated endocytosis in vitro. After a 4-h incubation, PNAs were largely localized in the nuclei of the cells; the mechanisms involved are still unclear. More than 70% inhibition of telomerase activity was observed when PNAs complementary to the RNA template of human telomerase were delivered to HepG2 cells using AF/DNA. The PNAs were stably associated with the AF/DNA conjugates in 50% serum at 37 °C for at least 3 h. The PNAs were spontaneously released from the conjugate through a strand exchange mechanism when complementary nucleic acid was added. The complexation of PNAs with the AF/DNA conjugates resulted in delivery of PNAs to liver after intravenous injection into mice. The present study indicates that conjugation to a natural proteinous ligand can be used as a non-toxic vector for cellular delivery of oligonucleotide analogs.

The structure of Yersinia pestis Caf1 polymer in free and adjuvant bound states

Caf1 of the plague bacterium, Yersinia pestis is a polymeric virulence factor and vaccine component, formed from monomers by a donor strand exchange (DSE) mechanism. Here, EM images of Caf1 reveal flexible polymers up to 1.5μm long (4MDa). The bead-like structures along the polymer are 5.8±1nm long and correspond to single Caf1 proteins. Short polymers often form circles, presumably by DSE. We also provide the first images of proteins bound to alhydrogel adjuvant. Caf1, hemocyanin and anthrax PA are all resolved clearly and Caf1 exhibits adjuvant bound stretches with long intervening loops draped from the edges.

Modeling the early stage of DNA sequence recognition within RecA nucleoprotein filaments

Homologous recombination is a fundamental process enabling the repair of double-strand breaks with a high degree of fidelity. In prokaryotes, it is carried out by RecA nucleofilaments formed on single-stranded DNA (ssDNA). These filaments incorporate genomic sequences that are homologous to the ssDNA and exchange the homologous strands. Due to the highly dynamic character of this process and its rapid propagation along the filament, the sequence recognition and strand exchange mechanism remains unknown at the structural level. The recently published structure of the RecA/DNA filament active for recombination (Chen et al., Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structure, Nature 2008, 453, 489) provides a starting point for new exploration of the system. Here, we investigate the possible geometries of association of the early encounter complex between RecA/ssDNA filament and double-stranded DNA (dsDNA). Due to the huge size of the system and its dense packing, we use a reduced representation for protein and DNA together with state-of-the-art molecular modeling methods, including systematic docking and virtual reality simulations. The results indicate that it is possible for the double-stranded DNA to access the RecA-bound ssDNA while initially retaining its Watson–Crick pairing. They emphasize the importance of RecA L2 loop mobility for both recognition and strand exchange.

The high-adhesive properties of the FimH adhesin of Salmonella enterica serovar Enteritidis are determined by a single F118S substitution

The binding properties of low- and high-adhesive forms of FimH adhesins from Salmonella enterica serovars Enteritidis and Typhimurium (S. Enteritidis and S. Typhimurium) were studied using chimeric proteins containing an additional peptide that represents an N-terminal extension of the FimF protein. This modification, by taking advantage of a donor strand exchange mechanism, closes the hydrophobic groove in the fimbrial domain of the FimH adhesin. Such self-complemented adhesins (scFimH) did not form aggregates and were more stable (resistant to proteolytic cleavage) than native FimH. High-adhesive variants of scFimH proteins, with alanine at position 61 and serine at position 118, were obtained by site-directed mutagenesis of fimH genes from low-adhesive variants of S. Enteritidis and S. Typhimurium, with glycine at position 61 and phenylalanine at position 118. Direct kinetic analysis using surface plasmon resonance (SPR) and glycoproteins carrying high-mannose carbohydrate chains (RNase B, horseradish peroxidase and mannan-BSA) revealed the existence of high- and low-adhesive allelic variants, not only in S. Typhimurium but also in S. Enteritidis. Using two additional mutants of low-adhesive FimH protein from S. Enteritidis (Gly61Ala and Phe118Ser), SPR analysis pointed to Ser118 as the major determinant of the high-adhesive phenotype of type 1 fimbriae from S. Enteritidis. These studies demonstrated for the first time that the functional differences observed with whole fimbriated bacteria could be reproduced at the level of purified adhesin. They strongly suggest that the adhesive properties of type 1 fimbriae are determined only by structural differences in the FimH proteins and are not influenced by the fimbrial shaft on which the adhesin is located.

Mechanism and Specificity of DNA Strand Exchange Catalyzed by Vaccinia DNA Topoisomerase Type I

The type I DNA topoisomerase from vaccinia virus (vTopo) forms a reversible covalent 3'-phosphotyrosyl linkage with a single strand of duplex DNA at the preferred sequence 5'-(C/T)CCTTp downward arrowN(-1)N(-2)N(-3)-3'. The enzyme-DNA covalent adduct is recombinogenic in cells, because the nicked strand downstream of the cleavage site can dissociate and be replaced by another DNA strand, potentially resulting in genome rearrangements if the enzyme executes strand ligation. Topo I could play an active role in strand exchange, either by altering the kinetics or thermodynamics of DNA strand binding or by serving as a proofreading gate to prevent ligation of incoming DNA strands containing mismatches. To address these questions, we have measured the kinetic and thermodynamic parameters for strand annealing to a purified vaccinia Topo I-DNA (vTopo-DNA) covalent complex containing a single-strand overhang and then compared them with the same overhang duplex in the absence of vTopo. We found that vTopo accelerates the strand association rate by 2-fold but has no effect on the rate of strand dissociation. vTopo has a similar small effect on the annealing parameters of a series of DNA strands containing single mismatches. In contrast, single base mismatches at the -1, -2, or -3 positions decreased the forward rate and equilibrium constant for reversible strand ligation by 10-fold. These data establish that while vTopo is a bystander during the annealing step of strand exchange, the enzyme strongly discriminates against mismatches close to the cleavage site during the subsequent events leading to strand ligation. A mechanism emerges where vTopo oscillates between an open state where the downstream DNA segment does not interact with the enzyme and a closed state where catalytically important contacts are formed with this region. This oscillation between an open and closed state of the covalently bound enzyme is likely important for regulating the number of DNA superhelical turns that are removed during the lifetime of the covalent complex with supercoiled substrates.

Mechanical Constraints on Hin Subunit Rotation Imposed by the Fis/Enhancer System and DNA Supercoiling during Site-Specific Recombination

Hin, a member of the serine family of site-specific recombinases, regulates gene expression by inverting a DNA segment. DNA inversion requires assembly of an invertasome complex in which a recombinational enhancer DNA segment bound by the Fis protein associates with the Hin synaptic complex at the base of a supercoiled DNA branch. Each of the four Hin subunits becomes covalently joined to the cleaved DNA ends, and DNA exchange occurs by translocation of a Hin subunit pair within the tetramer. We show here that, although the Hin tetramer forms a bidirectional molecular swivel, the Fis/enhancer system determines both the direction and number of subunit rotations. The chirality of supercoiling directs rotational direction, and the short DNA loop stabilized by Fis-Hin contacts limit rotational processivity, thereby ensuring that the DNA strands religate in the recombinant configuration. We identify multiple rotational conformers that are formed under different supercoiling and solution conditions.

DNA Strand Exchange on Liposome Surfaces

The mechanism of DNA strand exchange, performed in vivo by proteins in the Rec A family, is despite extensive studies not understood in full detail. We therefore want to study the molecular parameters involved in the exchange using an in vitro platform. Positively charged liposomes have been widely used as non-viral gene-carriers, where the positive charges attract the negatively charged DNA. We here show that DNA strand exchange is significantly enhanced, both in rate and yield, on the surface of cationic liposomes.We study the strand exchange reaction by adding an excess of non-labeled single strands to liposome bound DNA duplexes labeled with a FRET pair and monitor the exchange as an increase in donor emission. The fastest exchange is seen for liposomes containing 35% cationic lipid and when the positive charges on the outer leaflet of the liposomes equal the amount of negative charges on the added DNA. Both increasing and decreasing the amount of DNA slows down the exchange rate, either due to competition for the binding sites or due to that the single-strands on average are further away from the duplexes. By studying the exchange of mismatched DNA we conclude that the DNA opens in a “zipper-like” manner on the liposome surface, since a mismatched base-pair in the end of the sequence affects the exchange rate much more than a mismatch base-pair in the middle of the sequence. The liposome surface can be easily functionalized with lipids bearing specific functionalities and we intend to modify the surface to study the effects of hydrogen bonding and/or hydrophobic interactions.

Mechanism of DNA Strand Exchange at Liposome Surfaces Investigated Using Mismatched DNA

DNA strand exchange is of great importance in vivo for genetic recombination and DNA repair. The detailed mechanism of strand exchange is not understood in full detail despite extensive studies. Simplistic model systems in which molecular parameters can be varied independently are therefore of interest to study. We chose the surface of a positively charged liposome as a scaffold, which we recently demonstrated to be able to catalyze the exchange of fully complementary DNA oligonucleotides. We here study how single base pair mismatches affect the rate of strand exchange on the liposome surface. Interestingly, the rate of the exchange does not simply follow the stability of the duplex in solution, as determined by melting temperatures, but also depends sensitively on the position of the mismatch. For duplexes with similar melting temperatures, the exchange is much faster for a mismatch close to the end than for a mismatch in the middle of the sequence. Our results suggest that the single strands are stabilized by the liposome surface; therefore, the duplex is fraying more and the DNA opens up in a zipperlike fashion on the surface, increasing the probability of strand exchange. We also show that the competition between greater stability (higher Tm in solution) and higher concentration is important for the final composition of the duplex when a large excess of single strands is added to a complementary double-stranded DNA. Finally, the similar exchange rate constants for fully base-paired duplexes on the liposome surface when adding fully matched single strands or single strands with a mismatched base indicate that the rate is governed largely by separation of the initial duplex and not by the formation of the product duplex.

A chimeric Cre recombinase with regulated directionality

From bacterial viruses to humans, site-specific recombination and transposition are the major pathways for rearranging genomes on both long- and short-time scales. The site-specific pathways can be divided into 2 groups based on whether they are stochastic or regulated. Recombinases Cre and lambda Int are well-studied examples of each group, respectively. Both have been widely exploited as powerful and flexible tools for genetic engineering: Cre primarily in vivo and lambda Int primarily in vitro. Although Cre and Int use the same mechanism of DNA strand exchange, their respective reaction pathways are very different. Cre-mediated recombination is bidirectional, unregulated, does not require accessory proteins, and has a minimal symmetric DNA target. We show that when Cre is fused to the small N-terminal domain of Int, the resulting chimeric Cre recombines complex higher-order DNA targets comprising >200 bp encoding 16 protein-binding sites. This recombination requires the IHF protein, is unidirectional, and is regulated by the relative levels of the 3 accessory proteins, IHF, Xis, and Fis. In one direction, recombination depends on the Xis protein, and in the other direction it is inhibited by Xis. It is striking that regulated directionality and complexity can be conferred in a simple chimeric construction. We suggest that the relative ease of constructing a chimeric Cre with these properties may simulate the evolutionary interconversions responsible for the large variety of site-specific recombinases observed in Archaea, Eubacteria, and Eukarya.

Enhanced DNA strand exchange on positively charged liposomes

The mechanism of DNA strand exchange, performed in vivo by proteins in the RecA family, is, despite extensive studies, still not understood in detail. We are therefore investigating a model system, using the surface of a lipid vesicle, to systematically study the effect of individual parameters on the rate of the strand exchange reaction. DNA oligonucleotides are accumulated on the surface of cationic liposomes and the strand exchange is monitored using FRET. We show that in the presence of lipid vesicles containing 35% cationic lipid the strand exchange takes place with a significantly enhanced rate and improved yield, compared to in bulk without liposomes. The highest rate is observed at a charge ratio where the positive surface charge is matched by the negative charges from DNA, and thus all double- and single-stranded DNA can be bound to the liposome surface. The DNA duplex is destabilized on the positively charged surface and with the accumulation of complementary single strands in close proximity a fast exchange is facilitated. We also show that when aggregation of liposomes is minimized by incorporation of a PEG-modified lipid, the strand exchange is still efficient, although with a lower rate than for the unmodified system. We conclude that the liposome model system, with the possibility to systematically vary the surface composition, is a promising platform for further studies of DNA strand exchange reactions.

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 Å 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.