Visualization of RecA filaments and DNA by fluorescence microscopy

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.     

ATP-mediated conformational changes in the RecA filament

The crystal structure of the E. coli RecA protein was solved more than 10 years ago, but it has provided limited insight into the mechanism of homologous genetic recombination. Using electron microscopy, we have reconstructed five different states of RecA-DNA filaments. The C-terminal lobe of the RecA protein is modulated by the state of the distantly bound nucleotide, and this allosteric coupling can explain how mutations and truncations of this C-terminal lobe enhance RecA's activity. A model generated from these reconstructions shows that the nucleotide binding core is substantially rotated from its position in the RecA crystal filament, resulting in ATP binding between subunits. This simple rotation can explain the large cooperativity in ATP hydrolysis observed for RecA-DNA filaments.     

Lesion bypass by the Escherichia coli DNA polymerase V requires assembly of a RecA nucleoprotein filament

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATPgammaS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.     

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.     

Regulation of DNA strand exchange in homologous recombination

Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate.     

Two distinct conformational states define the interaction of human RAD51‐ATP with single‐stranded DNA

An essential mechanism for repairing DNA double‐strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single‐stranded DNA, promoting DNA‐strand exchange. Here, we study the interaction of hRAD51 with single‐stranded DNA using a single‐molecule approach. We show that ATP‐bound hRAD51 filaments can exist in two different states with different contour lengths and with a free‐energy difference of ~4 k_BT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly‐competent ADP‐bound configuration. In agreement with the single‐molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51‐ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51‐ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.     

Fluorescent human RAD51 reveals multiple nucleation sites and filament segments tightly associated along a single DNA molecule

The DNA strand-exchange reactions defining homologous recombination involve transient, nonuniform allosteric interactions between recombinase proteins and their DNA substrates. To study these mechanistic aspects of homologous recombination, we produced functional fluorescent human RAD51 recombinase and visualized recombinase interactions with single DNA molecules in both static and dynamic conditions. We observe that RAD51 nucleates filament formation at multiple sites on double-stranded DNA. This avid nucleation results in multiple RAD51 filament segments along a DNA molecule. Analysis of fluorescent filament patch size and filament kinks from scanning force microscopy (SFM) images indicate nucleation occurs minimally once every 500 bp. Filament segments did not rearrange along DNA, indicating tight association of the ATP-bound protein. The kinetics of filament disassembly was defined by activating ATP hydrolysis and following individual filaments in real time.     

Dynamics of bacteriophage T4 presynaptic filament assembly from extrinsic fluorescence measurements of Gp32-single-stranded DNA interactions

In the bacteriophage T4 homologous recombination system, presynaptic filament assembly on single-stranded (ssDNA) DNA requires UvsX recombinase, UvsY mediator, and Gp32 ssDNA-binding proteins. Gp32 exerts both positive and negative effects on filament assembly: positive by denaturing ssDNA secondary structure, and negative by competing with UvsX for ssDNA binding sites. UvsY is believed to help UvsX displace Gp32 from the ssDNA. To test this model we developed a real-time fluorescence assay for Gp32-ssDNA interactions during presynapsis, based on changes in the fluorescence of a 6-iodoacetamidofluorescein-Gp32 conjugate. Results demonstrate that the formation of UvsX presynaptic filaments progressively disrupts Gp32-ssDNA interactions. Under stringent salt conditions the disruption of Gp32-ssDNA by UvsX is both ATP- and UvsY-dependent. The displacement of Gp32 from ssDNA during presynapsis requires ATP binding, but not ATP hydrolysis, by UvsX protein. Likewise, UvsY-mediated presynapsis strongly requires UvsY-ssDNA interactions, and is optimal at a 1:1 stoichiometry of UvsY to UvsX and/or ssDNA binding sites. Presynaptic filaments formed in the presence of UvsY undergo assembly/collapse that is tightly coupled to the ATP hydrolytic cycle and to stringent competition for ssDNA binding sites between Gp32 and various nucleotide-liganded forms of UvsX. The data directly support the Gp32 displacement model of UvsY-mediated presynaptic filament assembly, and demonstrate that the transient induction of high affinity UvsX-ssDNA interactions by ATP are essential, although not sufficient, for Gp32 displacement. The underlying dynamics of protein-ssDNA interactions within presynaptic filaments suggests that rearrangements of UvsX, UvsY, and Gp32 proteins on ssDNA may be coupled to central processes in T4 recombination metabolism.     

Mechanism of presynaptic filament stabilization by the bacteriophage T4 UvsY recombination mediator protein

UvsY is the recombination mediator protein (RMP) of bacteriophage T4, which promotes homologous recombination by facilitating presynaptic filament assembly. The results of previous studies suggest that UvsY promotes the assembly of presynaptic filaments in part by stabilizing interactions between T4 UvsX recombinase and single-stranded DNA (ssDNA). To test this hypothesis, we studied the interactions of UvsX and UvsY with a fluorescein-derivatized oligonucleotide. This assay distinguishes between bipartite UvsX- or UvsY-ssDNA and tripartite UvsX-UvsY-ssDNA complex formation via differential fluorescence quenching effects. Salt stabilities of the three complexes were measured at equilibrium in the presence and absence of various nucleotide ligands of the UvsX protein and also under steady-state conditions for UvsX-catalyzed ssDNA-dependent ATP hydrolysis. The results demonstrate that UvsY globally stabilizes UvsX-ssDNA complexes, consistent with an increase in the apparent equilibrium binding affinity, K(ss)omega, of the UvsX-ssDNA interactions. The UvsY-mediated affinity increase is observed at equilibrium in the presence of ADP, ATPgammaS, or in the absence of the nucleotide and also at steady-state in the presence of ATP. Intriguingly, the stabilizing effects of UvsY and ATPgammaS on UvsX-ssDNA interactions are synergistic, indicating nonredundant mechanisms for UvsX-ssDNA complex stabilization by RMP versus nucleoside triphosphate effectors. Experiments with UvsY missense mutants defective in ssDNA binding demonstrate that UvsY-ssDNA interactions are of major importance in stabilizing UvsX-ssDNA complexes, whereas UvsY-UvsX protein-protein interactions provide residual stabilization energy. Together, the data is consistent with a mechanism in which UvsY stabilizes presynaptic filaments by organizing the ssDNA lattice into a structure that is favorable for UvsX-ssDNA interactions.     

Requirements for ATP binding and hydrolysis in RecA function in Escherichia coli

RecA is essential for recombination, DNA repair and SOS induction in Escherichia coli . ATP hydrolysis is known to be important for RecA's roles in recombination and DNA repair. In vitro reactions modelling SOS induction minimally require ssDNA and non-hydrolyzable ATP analogues. This predicts that ATP hydrolysis will not be required for SOS induction in vivo . The requirement of ATP binding and hydrolysis for SOS induction in vivo is tested here through the study of recA4159 (K72A) and recA2201 (K72R). RecA4159 is thought to have reduced affinity for ATP. RecA2201 binds, but does not hydrolyse ATP. Neither mutant was able to induce SOS expression after UV irradiation. RecA2201, unlike RecA4159, could form filaments on DNA and storage structures as measured with RecA–GFP. RecA2201 was able to form hybrid filaments and storage structures and was either recessive or dominant to RecA⁺, depending on the ratio of the two proteins. RecA4159 was unable to enter RecA⁺ filaments on DNA or storage structures and was recessive to RecA⁺. It is concluded that ATP hydrolysis is essential for SOS induction. It is proposed that ATP binding is essential for storage structure formation and ability to interact with other RecA proteins in a filament.     

Differential dynamic behavior of actin filaments containing tightly-bound Ca2+ or Mg2+ in the presence of myosin heads actively hydrolyzing ATP.

We have investigated how Ca2+ or Mg2+ bound at the high-affinity cation binding site in F-actin modulates the dynamic response of these filaments to ATP hydrolysis by attached myosin head fragments (S1). Rotational motions of the filaments were monitored using steady-state phosphorescence emission anisotropy of the triplet probe erythrosin-5-iodoacetamide covalently attached to cysteine 374 of actin. The anisotropy of filaments containing only Ca2+ increased from 0.080 to 0.137 upon binding S1 in a rigor complex and decreased to 0.065 in the presence of ATP, indicating that S1 induced additional rotational motions in the filament during ATP hydrolysis. The comparable anisotropy values for Mg(2+)-containing filaments were 0.067, 0.137, and 0.065, indicating that S1 hydrolysis did not induce measurable rotational motions in these filaments. Phalloidin, a fungal toxin which stabilizes F-actin and increases its rigidity, increased the anisotropy of F-actin containing either Ca2+ or Mg2+ but not the anisotropy of the 1:1 S1-actin complexes of these filaments. Mg(2+)-containing filaments with phalloidin bound also displayed increased rotational motions during S1 ATP hydrolysis. A strong positive correlation between the phosphorescence anisotropy of F-actin under specific conditions and the extent of the rotational motions induced by S1 during ATP hydrolysis suggested that the long axis torsional rigidity of F-actin plays a crucial role in modulating the dynamic response of the filaments to ATP hydrolysis by S1. Cooperative responses of F-actin to dynamic perturbations induced by S1 during ATP hydrolysis may thus be physically mediated by the torsional rigidity of the filament.     

Helical order in tarantula thick filaments requires the "closed" conformation of the myosin head

Myosin heads are helically ordered on the thick filament surface in relaxed muscle. In mammalian and avian filaments this helical arrangement is dependent on temperature and it has been suggested that helical order is related to ATP hydrolysis by the heads. To test this hypothesis, we have used electron microscopy and image analysis to study the ability and temperature dependence of analogs of ATP and ADP.Pi to induce helical order in tarantula thick filaments. ATP or analogs were added to rigor myofibrils or purified thick filaments at 22 degrees C and 4 degrees C and the samples negatively stained. The ADP.Pi analogs ADP.AlF4 and ADP.Vi, and the ATP analogs ADP.BeFx, AMPPNP and ATPgammaNH2, all induced helical order in tarantula thick filaments, independent of temperature. In the absence of nucleotide, or in the presence of ADP or the ATP analog, ATPgammaS, there was no helical ordering. According to crystallographic and tryptophan fluorescence studies, all of these analogs, except ATPgammaS and ADP, induce the "closed" conformation of the myosin head (in which the gamma phosphate pocket is closed). We suggest that helical order requires the closed conformation of the myosin head but is not dependent on the hydrolysis of ATP.     

Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination

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.     

Structure of helical RecA-DNA complexes. Complexes formed in the presence of ATP-gamma-S or ATP

Electron micrographs of RecA-DNA filaments, formed under several different conditions, have been analyzed and the filament images reconstructed in three dimensions. In the presence of ATP and a non-hydrolyzable ATP analog. ATP-gamma-S, the RecA protein forms with DNA a right-handed helical complex with a pitch of approximately 95 A. The most detailed view of the filament was obtained from analysis of RecA filaments on double-stranded DNA in the presence of ATP-gamma-S. There are approximately six subunits of RecA per turn of the helix, but both this number and the pitch are variable. From the examination of single filaments and filament-filament interactions, a picture of an extremely flexible protein structure emerges. The subunits of RecA protein are seen to be arranged in such a manner that the bound DNA must be partially exposed and able to come into contact with external DNA molecules. The RecA structure determined in the presence of ATP-gamma-S appears to be the same as the "pre-synaptic" state that occurs with ATP, in which there is recognition and pairing between homologous DNA molecules.     

Energetics of RecA-mediated recombination reactions. Without ATP hydrolysis RecA can mediate polar strand exchange but is unable to recycle

We demonstrate that the step of DNA strand exchange during RecA-mediated recombination reaction can occur equally efficiently in the presence or absence of ATP hydrolysis. The polarity of strand exchange is the same when instead of ATP its non-hydrolyzable analog adenosine-5'-O-(3-thiotriphosphate) is used. We show that the ATP dependence of recombination reaction is limited to the post-exchange stages of the reactions. The low DNA affinity state of RecA protomers, induced after ATP hydrolysis, is necessary for the dissociation of RecA-DNA complexes at the end of the reaction. This dissociation of RecA from DNA is necessary for the release of recombinant DNA molecules from the complexes formed with RecA and for the recycling of RecA protomers for another round of the recombination reaction.     

Functional Relationship of ATP Hydrolysis,Presynaptic Filament Stability,and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1

DMC1 and RAD51 are conserved recombinases that catalyze homologous recombination. DMC1 and RAD51 share similar properties in DNA binding, DNA-stimulated ATP hydrolysis, and catalysis of homologous DNA strand exchange. A large body of evidence indicates that attenuation of ATP hydrolysis leads to stabilization of the RAD51-ssDNA presynaptic filament and enhancement of DNA strand exchange. However, the functional relationship of ATPase activity, presynaptic filament stability, and DMC1-mediated homologous DNA strand exchange has remained largely unexplored. To address this important question, we have constructed several mutant variants of human DMC1 and characterized them biochemically to gain mechanistic insights. Two mutations, K132R and D223N, that change key residues in the Walker A and B nucleotide-binding motifs ablate ATP binding and render DMC1 inactive. On the other hand, the nucleotide-binding cap D317K mutant binds ATP normally but shows significantly attenuated ATPase activity and, accordingly, forms a highly stable presynaptic filament. Surprisingly, unlike RAD51, presynaptic filament stabilization achieved via ATP hydrolysis attenuation does not lead to any enhancement of DMC1-catalyzed homologous DNA pairing and strand exchange. This conclusion is further supported by examining wild-type DMC1 with non-hydrolyzable ATP analogues. Thus, our results reveal an important mechanistic difference between RAD51 and DMC1.     

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.     

Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament dissociation of RecA--DNA complex by inactive nucleotides.

The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA-DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA-DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5'-O-3-thiotriphosphate or guanosine 5'-O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA-DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex.     

RecA-promoted sliding of base pairs within DNA repeats: quantitative analysis by a slippage assay

RecA that catalyses efficient homology search and exchange of DNA bases has to effect major transitions in the structure as well as the dynamics of bases within RecA-DNA filament. RecA induces slippage of paired strands in poly(dA)-poly(dT) duplex using the energy of ATP hydrolysis. Here, we have adopted the targeted ligation assay and quantified the strand slippage within a short central cassette of (dA)(4)-(dT)(4) duplex. The design offers a stringent test case for scoring a cross-talk between A residues with those of T that are diagonally placed on the opposite strand at either -3, -2, -1, +1, +2, or +3 pairing frames. As expected, the cross-talk levels in RecA mediated as well as thermally annealed duplexes were maximal in non-diagonal pairing frame (i.e., 0-frame), the levels of which fell off gradually as the frames became more diagonal, i.e., -3<-2<-1 or +3<+2<+1. Interestingly, the level of cross-talk in naked duplexes was intrinsically less efficient in minus frames than their plus frame counterparts. The asymmetry created in naked duplexes by such a disparity between minus versus plus frames was partially obviated by RecA. Moreover, RecA promoted a significantly higher level of cross-talk selectively in -2 and -1 frames, as compared to that in naked DNA, which suggests a model that the elevated cross-talk in RecA filament may be limited to base pairs housed within the same rather than adjacent RecA monomers.     

Calorimetric analysis of binding of two consecutive DNA strands to RecA protein illuminates mechanism for recognition of homology

RecA protein recognises two complementary DNA strands for homologous recombination. To gain insight into the molecular mechanism, the thermodynamic parameters of the DNA binding have been characterised by isothermal calorimetry. Specifically, conformational changes of protein and DNA were searched for by measuring variations in enthalpy change (DeltaH) with temperature (heat capacity change, DeltaC(p)). In the presence of the ATP analogue ATPgammaS, the DeltaH for the binding of the first DNA strand depends upon temperature (large DeltaC(p)) and the type of buffer, in a way that is consistent with the organisation of disordered parts and the protonation of RecA upon complex formation. In contrast, the binding of the second DNA strand occurs without any pronounced DeltaC(p), indicating the absence of further reorganisation of the RecA-DNA filament. In agreement with these findings, a significant change in the CD spectrum of RecA was observed only upon the binding of the first DNA strand. In the absence of nucleotide cofactor, the DeltaH of DNA binding is almost independent of temperature, indicating a requirement for ATP in the reorganisation of RecA. When the second DNA strand is complementary to the first, the DeltaH is larger than that for non-complementary DNA strand, but less than the DeltaH of the annealing of the complementary DNA without RecA. This small DeltaH could reflect a weak binding that may facilitate the dissociation of only partly complementary DNA and thus speed the search for complementary DNA. The DeltaH of binding DNA sequences displaying strong base-base stacking is small for both the first and second binding DNA strand, suggesting that the second is also stretched upon interaction with RecA. These results support the proposal that the RecA protein restructures DNA, preparing it for the recognition of a complementary second DNA strand, and that the recognition is due mainly to direct base-base contacts between DNA strands.