DNA Strand Exchange Reaction Research Articles

Biochemical characterization of Borrelia burgdorferi's RecA protein.

RecA plays key roles in DNA recombination, replication and repair. Mutation of recA in the Lyme disease spirochete, Borrelia burgdorferi, fails to produce some of the phenotypes expected from study of recA mutation in other organisms. ‘Missing’ recA phenotypes include a lack of growth or viability effects, including in the presence of DNA damage, and a lack of a role in vlsE antigenic variation and infectivity. We present a purification and biochemical characterization of recombinant B. burgdorferi RecA protein. We find that B. burgdorferi RecA displays the expected properties of being a DNA-dependent ATPase, of having an intrinsic binding preference for ssDNA over dsDNA enhanced by ATP binding, of promoting DNA pairing and strand exchange reactions and of having a detectable coprotease activity with E. coli LexA repressor. DNA pairing and strand exchange reactions promoted by B. burgdorferi RecA show an unusually strong dependence upon the presence of the cognate ssDNA binding protein (SSB) but are very sensitive to inhibition by SSB when the ssDNA was prebound by SSB. This indicates B. burgdorferi RecA may have an enhanced requirement for recombinational mediators to promote RecA-SSB exchange, despite the absence of homologues of the RecF pathway proteins that normally play this role in eubacteria. Finally, we do not find any unusual, intrinsic properties of B. burgdorferi’s RecA protein to explain the unusual phenotype of recA mutation and suggest that there may be alternative recombinase functions that could explain the ‘missing’ phenotypes.

Abstract 2474: The mechanistic role of polyamines in DNA double-strand break repair

Abstract Polyamines are essential organic cations for cell growth, proliferation and tissue remodeling. The rate-limiting step in the biosynthesis of polyamines is catalyzed by ornithine decarboxylase (ODC). The activity of ODC and the level of intracellular polyamines significantly increase in many cancers, indicating the critical role of polyamines in tumorigenesis. Depletion of polyamines sensitizes cells to DNA damage agents that lead to DNA double-strand breaks (DSBs), suggesting that endogenous polyamines may participate in DNA repair process. However, the mechanistic role of polyamines in DSB repair remains largely unknown. Here, we wish to ascertain the role of polyamines in DSBs repair pathway and to delineate the underlying mechanism by combining biochemical analyses, cell-based and animal approaches. Our cell-based experiments demonstrated that polyamines indeed participate in DNA double-strand break repair. Specifically, the level of intracellular polyamines regulates homologous recombination (HR) repair pathway rather than non-homologous end joining (NHEJ). Furthermore, our in vitro reconstitution system and functional analyses demonstrated that polyamines significantly enhance RAD51-mediated DNA strand exchange reaction. Importantly, we reveal that the stimulatory effect of polyamines by RAD51 stems from the enhancement of duplex DNA captures in the DNA exchange reaction. Finally, the physiological role of polyamines in DSBs repair was examined by using mouse hair follicle as a model system. The animal model clearly highlights the significant contribution of polyamines to the DNA repair in vivo. Our findings thus furnish valuable insight into the mechanistic basis of polyamines in DNA repair. Citation Format: Chih-Ying Lee, Guan-Chin Su, Min-Yu Ko, Wen-Yen Huang, Geen-Dong Chang, Sung-Jan Lin, Peter Chi. The mechanistic role of polyamines in DNA double-strand break repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2474. doi:10.1158/1538-7445.AM2017-2474

Exploring the Role of ATP Hydrolysis in the RecA Protein via Stalled DNA Intermediates

A central participant in the recombinational DNA‐repair system is the recombinase protein, RecA, which acts as a DNA pairing enzyme that is responsible for promoting an exchange of homologous DNA strands. Physical studies have revealed three DNA binding sites buried within the interior of the RecA filament. This current understanding of RecA is incomplete as it fails to explain how a four‐strand DNA exchange occurs between two duplex DNA fragments.The bacterial RecA homologue is unique in that it couples ATP‐hydrolysis with DNA strand exchange. ATP hydrolysis occurrs uniformly throughout the RecA filament, with only a small fraction of ATP used for filament disassembly. The mechanism by which the RecA filament utilizes the rest of ATP hydrolysis to perform DNA strand exchange reactions is not fully understood. My aim is to explore the hypothesis that the majority of ATP utilized by the reaction is hydrolyzed at binding sites located longitudinally along both the interior and exterior of the filament, providing the energy needed to rotate one segment of DNA around another.This will be explored by performing RecA mediated strand‐exchange reactions with a variety of linear, double stranded DNA substrates containing heterologous regions. When the RecA protein is run into a heterologous barrier, a stalled DNA‐protein intermediate molecule is the product. Formation of these intermediates has been obtained. Imaging with Electron Microscopy shows topological changes that offer fundamental insight into the universal mechanics of RecA‐mediated strand exchanges. Furthermore, evidence shows that introduction of the protein Uup modifies the structure of the intermediates, providing insight into Uup's function.

Antibody-controlled actuation of DNA-based molecular circuits

DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody–epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification.

Shape changing thin films powered by DNA hybridization.

Active materials that respond to physical and chemical stimuli can be used to build dynamic micromachines that lie at the interface between biological systems and engineered devices. In principle, the specific hybridization of DNA can be used to form a library of independent, chemically driven actuators for use in such microrobotic applications and could lead to device capabilities that are not possible with polymer- or metal-layer-based approaches. Here, we report shape changing films that are powered by DNA strand exchange reactions with two different domains that can respond to distinct chemical signals. The films are formed from DNA-grafted gold nanoparticles using a layer-by-layer deposition process. Films consisting of an active and a passive layer show rapid, reversible curling in response to stimulus DNA strands added to solution. Films consisting of two independently addressable active layers display a complex suite of repeatable transformations, involving eight mechanochemical states and incorporating self-righting behaviour.

ATP hydrolysis Promotes Duplex DNA Release by the RecA Presynaptic Complex

Homologous recombination is an important DNA repair pathway that plays key roles in maintaining genome stability. Escherichia coli RecA is an ATP-dependent DNA-binding protein that catalyzes the DNA strand exchange reactions in homologous recombination. RecA assembles into long helical filaments on single-stranded DNA, and these presynaptic complexes are responsible for locating and pairing with a homologous duplex DNA. Recent single molecule studies have provided new insights into RecA behavior, but the potential influence of ATP in the reactions remains poorly understood. Here we examine how ATP influences the ability of the RecA presynaptic complex to interact with homologous dsDNA. We demonstrate that over short time regimes, RecA presynaptic complexes sample heterologous dsDNA similarly in the presence of either ATP or ATPγS, suggesting that initial interactions do not depend on ATP hydrolysis. In addition, RecA stabilizes pairing intermediates in three-base steps, and stepping energetics is seemingly unaltered in the presence of ATP. However, the overall dissociation rate of these paired intermediates with ATP is ∼4-fold higher than with ATPγS. These experiments suggest that ATP plays an unanticipated role in promoting the turnover of captured duplex DNA intermediates as RecA attempts to align homologous sequences during the early stages of recombination.

Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics

The DNA strand exchange protein RAD51 facilitates the central step in homologous recombination, a process fundamentally important for accurate repair of damaged chromosomes, restart of collapsed replication forks, and telomere maintenance. The active form of RAD51 is a nucleoprotein filament that assembles on single-stranded DNA (ssDNA) at the sites of DNA damage. The c-Abl tyrosine kinase and its oncogenic counterpart BCR-ABL fusion kinase phosphorylate human RAD51 on tyrosine residues 54 and 315. We combined biochemical reconstitutions of the DNA strand exchange reactions with total internal reflection fluorescence microscopy to determine how the two phosphorylation events affect the biochemical activities of human RAD51 and properties of the RAD51 nucleoprotein filament. By mimicking RAD51 tyrosine phosphorylation with a nonnatural amino acid, p-carboxymethyl-l-phenylalanine (pCMF), we demonstrated that Y54 phosphorylation enhances the RAD51 recombinase activity by at least two different mechanisms, modifies the RAD51 nucleoprotein filament formation, and allows RAD51 to compete efficiently with ssDNA binding protein RPA. In contrast, Y315 phosphorylation has little effect on the RAD51 activities. Based on our work and previous cellular studies, we propose a mechanism underlying RAD51 activation by c-Abl/BCR-ABL kinases.

Roleof the RAD51-SWI5-SFR1 Ensemble in homologous recombination.

During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5–SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of ADP release. In this study, we have strived to determine the basis for physical and functional interactions between Mus musculus SWI5–SFR1 and RAD51. We found that SWI5–SFR1 preferentially associates with the oligomeric form of RAD51. Specifically, a C-terminal domain within SWI5 contributes to RAD51 interaction. With specific RAD51 interaction defective mutants of SWI5–SFR1 that we have isolated, we show that the physical interaction is indispensable for the stimulation of the recombinase activity of RAD51. Our results thus help establish the functional relevance of the trimeric RAD51–SWI5–SFR1 complex and provide insights into the mechanistic underpinnings of homology-directed DNA repair in mammalian cells.

Detecting 5-methylcytosine using an enzyme-free DNA strand exchange reaction without pretreatment under physiological conditions.

We present here a novel and efficient method for 5mC detection using a DNA strand exchange reaction (SER) strategy. This enzyme-free method needs no pre-treatment of target DNAs and can be adapted to most of the target duplexes under physiological conditions. The high sequence selectivity of this method can distinguish 5mC from normal cytosine in an accurate manner.

Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase.

Human RAD51 protein catalyzes DNA pairing and strand exchange reactions that are central to homologous recombination and homology-directed DNA repair. Successful recombination/repair requires the formation of a presynaptic filament of RAD51 on ssDNA. Mutations in BRCA2 and other proteins that control RAD51 activity are associated with human cancer. Here we describe a set of mutations associated with human breast tumors that occur in a common structural motif of RAD51. Tumor-associated D149N, R150Q and G151D mutations map to a Schellman loop motif located on the surface of the RecA homology domain of RAD51. All three variants are proficient in DNA strand exchange, but G151D is slightly more sensitive to salt than wild-type (WT). Both G151D and R150Q exhibit markedly lower catalytic efficiency for adenosine triphosphate hydrolysis compared to WT. All three mutations alter the physical properties of RAD51 nucleoprotein filaments, with G151D showing the most dramatic changes. G151D forms mixed nucleoprotein filaments with WT RAD51 that have intermediate properties compared to unmixed filaments. These findings raise the possibility that mutations in RAD51 itself may contribute to genome instability in tumor cells, either directly through changes in recombinase properties, or indirectly through changes in interactions with regulatory proteins.

DNA strand exchange and RecA homologs in meiosis.

Homology search and DNA strand-exchange reactions are central to homologous recombination in meiosis. During meiosis, these processes are regulated such that the probability of choosing a homolog chromatid as recombination partner is enhanced relative to that of choosing a sister chromatid. This regulatory process occurs as homologous chromosomes pair in preparation for assembly of the synaptonemal complex. Two strand-exchange proteins, Rad51 and Dmc1, cooperate in regulated homology search and strand exchange in most organisms. Here, we summarize studies on the properties of these two proteins and their accessory factors. In addition, we review current models for the assembly of meiotic strand-exchange complexes and the possible mechanisms through which the interhomolog bias of recombination partner choice is achieved.

Mechanism of mycobacterial recombination mediator proteins function in homologous recombination and strand annealing (735.12)

Recombination mediator proteins (RMPs) are important for genome stability in all organisms and support two reactions: the recombinase binding to single‐stranded (ss)DNA to initiate homologous recombination (HR) and strand annealing. Both reactions are inhibited by ssDNA binding proteins (e.g. gp32, SSB, RPA). RMPs promote DNA transfer from SSB to recombinase or to anneal DNA. In spite of two ubiquitous functions, RMPs lack sequence and structural homology. Previously, we demonstrated that E. coli RMPs, RecOR, do not dismiss SSB from ssDNA, but form the complex with SSB altering the conformation of bound ssDNA. RecO binds SSB and the complex promotes DNA annealing or RecA loading in the absence or presence of RecR, respectively. Surprisingly, mycobacterial RecO does not interact with SSB. Yet, both proteins form complex on the shared ssDNA substrate in both pathways.In mycobacteria, RecFOR deletion reduces clastogens resistance to the same degree as a deletion of the major homologous recombinase RecA. Chromosomal DNA break repair assay surprisingly revealed that all three proteins are involved in both pathways: HR and annealing. Here we will describe biochemical studies of DNA annealing and strand exchange reactions supported by mycobacterial RecFOR. The selection of one or the other pathway is dictated by the availability of recombinase or complimentary strand and the stoichiometry of the DNA‐protein complexes. We believe that the intermediate complex of RMP with SSB and ssDNA represents a common critical step in reactions supported by all RMPs during HR and strand annealing DNA repair pathways in all organisms.

DNA strand exchange reaction activated by cationic comb-type copolymers having ureido groups.

Strand exchange reactions (SERs) of nucleic acids are essential for genetic recombination and often require nucleic acid chaperone activity. A variety of proteins induce strand exchange reactions in vivo. We have reported that cationic comb-type copolymers composed of a polycation backbone and hydrophilic graft chains greatly accelerate the SER in vitro and exhibit nucleic acid chaperone like activity. To elucidate structure/function relationships involved in the SER accelerating activity of the copolymer, we modified the copolymer with ureido groups. The melting temperature of dsDNA in the presence of the copolymers with ureido groups decreased with increasing ureido content, suggesting that the ureido groups have a destabilizing effect on the DNA duplex. Copolymers modified with ureido groups (about 50 mol%) accelerated a SER more strongly than unmodified copolymers. Given our previous observation that modification of the copolymer with guanidino groups resulted in enhanced chaperone-like activity, we propose that incorporation of chaotropic functional groups into the copolymer structure results in an increase in nucleic acid chaperone-like activity.

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.

Kinetics of Presynaptic Filament Assembly in the Presence of Single-Stranded DNA Binding Protein and Recombination Mediator Protein

Enzymes of the RecA/Rad51 family catalyze DNA strand exchange reactions that are important for homologous recombination and for the accurate repair of DNA double-strand breaks. RecA/Rad51 recombinases are activated by their assembly into presynaptic filaments on single-stranded DNA (ssDNA), a process that is regulated by ssDNA binding protein (SSB) and mediator proteins. Mediator proteins stimulate strand exchange by accelerating the rate-limiting displacement of SSB from ssDNA by the incoming recombinase. The use of mediators is a highly conserved strategy in recombination, but the precise mechanism of mediator activity is unknown. In this study, the well-defined bacteriophage T4 recombination system (UvsX recombinase, Gp32 SSB, and UvsY mediator) is used to examine the kinetics of presynaptic filament assembly on native ssDNA in vitro. Results indicate that the ATP-dependent assembly of UvsX presynaptic filaments on Gp32-covered ssDNA is limited by a salt-sensitive nucleation step in the absence of mediator. Filament nucleation is selectively enhanced and rendered salt-resistant by mediator protein UvsY, which appears to stabilize a prenucleation complex. This mechanism potentially explains how UvsY promotes presynaptic filament assembly at physiologically relevant ionic strengths and Gp32 concentrations. Other data suggest that presynaptic filament assembly involves multiple nucleation events, resulting in many short UvsX-ssDNA filaments or clusters, which may be the relevant form for recombination in vivo. Together, these findings provide the first detailed kinetic model for presynaptic filament assembly involving all three major protein components (recombinase, mediator, and SSB) on native ssDNA.

The telomere resolvase of the Lyme disease spirochete, Borrelia burgdorferi, promotes DNA single-strand annealing and strand exchange

Spirochetes of the genus Borrelia include the tick-transmitted causative agents of Lyme disease and relapsing fever. They possess unusual genomes composed mainly of linear replicons terminated by closed DNA hairpin telomeres. Hairpin telomeres present an uninterrupted DNA chain to the replication machinery overcoming the ‘end-replication problem’ for the linear replicons. Hairpin telomeres are formed from inverted repeat replicated telomere junctions by the telomere resolvase, ResT. ResT uses a reaction mechanism similar to that of the type IB topoisomerases and tyrosine recombinases. We report here that ResT also possesses single-strand annealing activity and a limited ability to promote DNA strand exchange reactions on partial duplex substrates. This combination of activities suggests ResT is a nexus between the seemingly distinct processes of telomere resolution and homologous recombination. Implications for hairpin telomere replication and linear plasmid recombination, including antigenic variation, are discussed.

Opposing Roles for Two Molecular Forms of Replication Protein A in Rad51-Rad54-Mediated DNA Recombination in Plasmodium falciparum

ABSTRACTThe bacterial RecA protein and its eukaryotic homologue Rad51 play a central role in the homologous DNA strand exchange reaction during recombination and DNA repair. Previously, our lab has shown that PfRad51, the Plasmodium falciparum homologue of Rad51, exhibited ATPase activity and promoted DNA strand exchange in vitro. In this study, we evaluated the catalytic functions of PfRad51 in the presence of putative interacting partners, especially P. falciparum homologues of Rad54 and replication protein A. PfRad54 accelerated PfRad51-mediated pairing between single-stranded DNA (ssDNA) and its homologous linear double-stranded DNA (dsDNA) in the presence of 0.5 mM CaCl2. We also present evidence that recombinant PfRPA1L protein serves the function of the bacterial homologue single-stranded binding protein (SSB) in initiating homologous pairing and strand exchange activity. More importantly, the function of PfRPA1L was negatively regulated in a dose-dependent manner by PfRPA1S, another RPA homologue in P. falciparum. Finally, we present in vivo evidence through comet assays for methyl methane sulfonate-induced DNA damage in malaria parasites and accompanying upregulation of PfRad51, PfRad54, PfRPA1L, and PfRPA1S at the level of transcript and protein needed to repair DNA damage. This study provides new insights into the role of putative Rad51-interacting proteins involved in homologous recombination and emphasizes the physiological role of DNA damage repair during the growth of parasites.

Equilibrious Strand Exchange Promoted by DNA Conformational Switching

Most of DNA strand exchange reactions in vitro are based on toehold strategy which is generally nonequilibrium, and intracellular strand exchange mediated by proteins shows little sequence specificity. Herein, a new strand exchange promoted by equilibrious DNA conformational switching is verified. Duplexes containing c-myc sequence which is potentially converted into G-quadruplex are designed in this strategy. The dynamic equilibrium between duplex and G4-DNA is response to the specific exchange of homologous single-stranded DNA (ssDNA). The SER is enzyme free and sequence specific. No ATP is needed and the displaced ssDNAs are identical to the homologous ssDNAs. The SER products and exchange kenetics are analyzed by PAGE and the RecA mediated SER is performed as the contrast. This SER is a new feature of G4-DNAs and a novel strategy to utilize the dynamic equilibrium of DNA conformations.

128 Some derivatives of 1,3-diazaadamantane strongly stimulate strand exchange reaction between short oligonucleotides

Earlier, we characterized the behavior of HG122 (compound III) in the DNA Strand Exchange Reaction (SER) and found that 1,3-diazaadamantane derivative facilitates SER in the system of short oligonucleotides (Gabrielian et al., 2011). In the present study, a series of new derivatives of 1,3-diazaadamantane have been synthesized with the purpose to discern how small variations in the compound structure can influence its activity in SER and try to get more effective substances for stimulation of SER. We revealed that most of the small variations in the structure significantly influenced the compounds’ efficacy in accelerating SER. For example, an increase in the compounds’ aliphatic chain lengths considerably enhanced its efficiency in SER stimulation and in the series of compounds presented in the Figure HG188 (compound IV) was eminently the most potent agent in the stimulation of SER. Small modifications in other parts of the 1,3-diazaadamantane molecule also influenced the SER stimulation and several derivatives more efficient in facilitating SER than HG122 were revealed. Some of the compounds exhibited virtually negligible capability to stimulate SER but, interestingly, out of 12 derivatives characterized, agents that retard spontaneous SER were not found. Earlier, the stimulation of the DNA strand exchange was documented for different ligands of the policationic nature such as Cationic Comb Copolymers (Kim et al., 2003), linker histones (Bocharova et al., 2012), cobalt hexamine (unpublished observation) etc. The present results provide us with a novel class of SER facilitating compounds – the cationic amphiphiles that can serve as interesting objects for further understanding of different aspects of DNA SER. Biomedical implications and prospects in biomedical applications of these and similar compounds’ activity in SER remain to be investigated.

Characterization of Multi-Functional Properties and Conformational Analysis of MutS2 from Thermotoga maritima MSB8

The MutS2 homologues have received attention because of their unusual activities that differ from those of MutS. In this work, we report on the functional characteristics and conformational diversities of Thermotoga maritima MutS2 (TmMutS2). Various biochemical features of the protein were demonstrated via diverse techniques such as scanning probe microscopy (SPM), ATPase assays, analytical ultracentrifugation, DNA binding assays, size chromatography, and limited proteolytic analysis. Dimeric TmMutS2 showed the temperature-dependent ATPase activity. The non-specific nicking endonuclease activities of TmMutS2 were inactivated in the presence of nonhydrolytic ATP (ADPnP) and enhanced by the addition of TmMutL. In addition, TmMutS2 suppressed the TmRecA-mediated DNA strand exchange reaction in a TmMutL-dependent manner. We also demonstrated that small-angle X-ray scattering (SAXS) analysis of dimeric TmMutS2 exhibited nucleotide- and DNA-dependent conformational transitions. Particularly, TmMutS2-ADPnP showed the most compressed form rather than apo-TmMutS2 and the TmMutS2-ADP complex, in accordance with the results of biochemical assays. In the case of the DNA-binding complexes, the stretched conformation appeared in the TmMutS2-four-way junction (FWJ)-DNA complex. Convergences of biochemical- and SAXS analysis provided abundant information for TmMutS2 and clarified ambiguous experimental results.