Activation of recA protein. The open helix model for LexA cleavage.

RecA protein is induced by the binding of DNA and ATP to become active in the hydrolysis of ATP and the cleavage of repressors. These reactions appear to depend on the structural state of the protein polymerized along the DNA, i.e. a helical coat of six RecA per turn of 95 to 100 A pitch. In support of this model of the active conformation, it was shown that high concentrations of salt also induce this helical polymerized state as well as the enzymatic activities. Here, we describe that, in vitro and with the non-hydrolyzable analogue ATP gamma S, RNA and heparin can also induce both the structural transition and the enzymatic activation of RecA to LexA cleavage in accordance with the model. RNA and heparin do not support the reaction in the presence of ATP, and they do not induce the hydrolysis of ATP either, suggesting that, in contrast to ATP gamma S, the nucleotide is not bound stably enough, and that the combined affinities of polynucleotide and ATP actually modulate the discrimination of RecA for the various possible inducers in vivo.     

Purification, crystallization and preliminary X-ray diffraction studies of the bacteriophage φ29 connector particle

The connector or portal particle from double-stranded DNA bacteriophage φ29 has been crystallized. This structure, which connects the head of the virus with the tail and plays a central role in prohead assembly and DNA packaging and translocation, is formed by 12 subunits of the p10 protein and has a molecular weight of 430 kDa. The connector structure was proteolysed with endoproteinase Glu-C from Staphylococcus aureus V8, which removes 13 and 18 amino acids from the amino- and carboxy-terminal regions of the p10 protein, respectively. Two crystal forms were grown from drops containing an alcohol solution and paraffin oil. Crystals of form I are monoclinic, space group C2 with cell dimensions a=416.86 Å, b=227.62 Å, c=236.68 Å and β=96.3° and contain four connector particles per asymmetric unit. Crystals of form II are tetragonal, space group P4₂2₁2 with cell dimensions a=b=170.2 Å, c=156.9 Å and contain half a particle per asymmetric unit. X-ray diffraction data from both native crystal forms have been collected to 6.0 and 3.2 Å respectively, using synchrotron radiation. Crystals of form II are likely to have the same packing arrangement as the two-dimensional crystals analyzed previously by electron microscopy.     

Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible.

We have used electron microscopy to examine the two major conformational states of the helical filament formed by the RecA protein of Escherichia coli. The compressed filament, formed in the absence of a nucleotide cofactor either as a self-polymer or on a single-stranded DNA molecule, is characterized in solution by about 6.1 subunits per turn of a 76 A pitch helix, and appears to be inactive with respect to all RecA activity. The active state of the filament, formed with ATP or an ATP analog on either a single or double-stranded DNA substrate, has about 6.2 subunits per turn of a 94 A pitch helix. Measurements of the contour length of RecA-covered single-stranded DNA circles in ice, formed in the absence of nucleotide cofactor, indicate that each RecA subunit binds five bases, in contrast to the three bases or base-pairs per subunit in the active state. The different stoichiometries of DNA binding suggests that the two polymeric forms are not interconvertible, as has been suggested on biochemical grounds. A three-dimensional reconstruction of the inactive state shows the same general features as the 83 A pitch filament present in the RecA crystal. This structural similarity and the fact that the crystal does not contain ATP or DNA suggests that the crystal structure is more similar to the compressed filament than the active, extended filament.     

The pairing activity of stable nucleoprotein filaments made from recA protein, single-stranded DNA, and adenosine 5'-(gamma-thio)triphosphate

Under conditions that diminish secondary structure in single-stranded DNA, stable presynaptic filaments can be formed by recA protein in the presence of the nonhydrolyzable analog ATP gamma S, without the need for Escherichia coli single strand binding protein. Such stable presynaptic filaments resemble those formed in the presence of ATP and pair efficiently with homologous duplex DNA. Since this kind of stable filament does not displace a strand from the duplex molecule, it provides a model substrate to study synapsis independent of the earlier and later stages of the recA reaction. Even though detectable strand displacement did not occur in the presence of ATP gamma S, both single strand and double strand breaks in duplex DNA stimulated homologous pairing. These and related observations support the view that the presynaptic nucleoprotein filament and naked duplex DNA intertwine to form a nascent joint in which the duplex DNA is partially unwound, i.e. in which the pitch of the involved duplex segment is reduced.     

Inhibition of recA protein promoted ATP hydrolysis. 1. ATP gamma S and ADP are antagonistic inhibitors

ADP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) inhibit recA protein promoted ATP hydrolysis by fundamentally different mechanisms. In both cases, at least two modes of inhibition are observed. For ADP, the first mode is competitive inhibition. The second mode is manifested by dissociation of recA protein from DNA. These are readily distinguished in a comparison of ATP hydrolyses that are activated by (a) DNA and (b) high (approximately 2 M) salt concentrations. Competitive inhibition with a significant degree of cooperativity is observed under both sets of conditions, although the DNA-dependent activity is more sensitive to ADP than the high-salt reaction. The reaction in the presence of poly(deoxythymidylic acid) or duplex DNA ceases when about 60% of the available ATP is hydrolyzed, reflecting an ADP-mediated dissociation of recA protein from the DNA that is governed by the ADP/ATP ratio. In contrast, ATP hydrolysis proceeds nearly to completion at high salt concentrations. At high concentrations of ATP and ATP gamma S, ATP gamma S also acts as a competitive inhibitor. At low concentrations of ATP gamma S and ATP, however, ATP gamma S activates ATP hydrolysis. These patterns are observed for recA-mediated ATP hydrolysis with either high salt concentrations or a poly(deoxythymidylic acid) [poly(dT)] cofactor, although the activation is observed at much lower ATP and ATP gamma S concentrations when poly(dT) is used. ATP gamma S can also relieve the inhibitory effect of ADP under some conditions. ATP gamma S and ADP are antagonistic inhibitors, reinforcing the idea that they stabilize different conformations of the protein and suggesting that these conformations are mutually exclusive. The ATP gamma S (ATP) conformation is active in ATP hydrolysis. The ADP conformation is inactive.     

The inactive form of recA protein: the 'compact' structure.

When recA protein is enzymatically inactive in vitro, it adopts a more compact helical polymer form than that of the active protein polymerized onto DNA in the presence of ATP. Here we describe some aspects of this structure. By cryo-electron microscopy, a pitch of 76 A is found for both the self-polymer and the inactive complex with ssDNA. A smaller pitch of 64 A is observed in conventional electron micrographs. The contour length of complexes with ssDNA was used to estimate the binding stoichiometry in the compact complex, 6 +/- 1 nt/recA. In addition, the compact structure was observed in vivo in Escherichia coli: inclusion bodies produced upon induction of recA expression in an overproducing strain have a fibrous morphology with the structural parameters of the compact polymer.     

Dissociation pathway for recA nucleoprotein filaments formed on linear duplex DNA

recA protein forms stable filaments on duplex DNA at low pH. When the pH is shifted above 6.8, recA protein remains stably bound to nicked circular DNA, but not to linear DNA. Dissociation of recA protein from linear duplex DNA proceeds to a non-zero endpoint. The kinetics and final extent of dissociation vary with several experimental parameters. The instability on linear DNA is most readily explained by a progressive unidirectional dissociation of recA protein from one end of the filament. Dissociation of recA protein from random points in the filament is eliminated as a possible mechanism by several observations: (1) the requirement for a free end; (2) the inverse and linear dependence of the rate of dissociation on DNA length (at constant DNA base-pair concentration); and (3) the kinetics of exposure of a restriction endonuclease site in the middle of the DNA. Evidence against another possible mechanism, ATP-mediated translocation of the filament along the DNA, is provided by a novel effect of the non-hydrolyzable ATP analog, ATP gamma S, which generally induces recA protein to bind any DNA tightly and completely inhibits ATP hydrolysis. We find that very low, sub-saturating levels of ATP gamma S completely stabilize the filament, while most of the ATP hydrolysis continues. If these levels of ATP gamma S are introduced after dissociation has commenced, further dissociation is blocked, but re-association does not occur. These observations are inconsistent with movement of recA protein along DNA that is tightly coupled to ATP hydrolysis. The recA nucleoprotein filament is polar and the protein binds the two strands asymmetrically, polymerizing mainly in the 5' to 3' direction on the initiating strand of a single-stranded DNA tailed duplex molecule. A model consistent with these results is presented.     

A RecA Protein Surface Required for Activation of DNA Polymerase V

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3''-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3''-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA''₂B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis.     

Actin paracrystal in the acrosome of newt sperm

When a newt sperm-head was treated with trypsin and DNase, an arrow-like thin rod was revealed. This rod presumably corresponds to the ‘perforatorium’ described by Picheral [1, 2] in Pleurodele sperm. It consisted of one apical and one caudal part. In the apical part there appeared to be an envelope with a 530 Å structural repeat, inside which coursed a filament bundle, presumably identical with that in the caudal part. In the caudal part, a characteristic filament bundle, quite similar to the paracrystal of rabbit skeletal actin [3], was observed after extensive treatment with trypsin. The optical diffraction pattern of this bundle indicates that it has the same helical symmetry as that of rabbit skeletal actin [4] but slightly different from that of the acrosomal process of Limulus sperm [5]. The diffraction pattern frequently has a strong meridional reflection at about (27 Å)⁻¹, which is usually observed only with low intensity in the actin paracrystals. This fact suggests that the structural unit in the bundle has a shape considerably different from that of the usual G-actin.     

Role of the conserved lysine within the Walker A motif of human DMC1

During meiosis, the RAD51 recombinase and its meiosis-specific homolog DMC1 mediate DNA strand exchange between homologous chromosomes. The proteins form a right-handed nucleoprotein complex on ssDNA called the presynaptic filament. In an ATP-dependent manner, the presynaptic filament searches for homology to form a physical connection with the homologous chromosome. We constructed two variants of hDMC1 altering the conserved lysine residue of the Walker A motif to arginine (hDMC1_K132R) or alanine (hDMC1_K132A). The hDMC1 variants were expressed in Escherichia coli and purified to near homogeneity. Both hDMC1_K132R and hDMC1_K132A variants were devoid of ATP hydrolysis. The hDMC1_K132R variant was attenuated for ATP binding that was partially restored by the addition of either ssDNA or calcium. The hDMC1_K132R variant was partially capable of homologous DNA pairing and strand exchange in the presence of calcium and protecting DNA from a nuclease, while the hDMC1_K132A variant was inactive. These results suggest that the conserved lysine of the Walker A motif in hDMC1 plays a key role in ATP binding. Furthermore, the binding of calcium and ssDNA promotes a conformational change in the ATP binding pocket of hDMC1 that promotes ATP binding. Our results provide evidence that the conserved lysine in the Walker A motif of hDMC1 is critical for ATP binding which is required for presynaptic filament formation.     

Enhanced recA protein binding to Z DNA represents a kinetic perturbation of a general duplex DNA binding pathway

recA protein binding to duplex DNA is enhanced when a B form DNA substrate is replaced with a left-handed Z form helix. This represents a kinetic rather than an equilibrium effect. Binding to Z DNA is much faster than binding to B DNA. In other respects, binding to the two DNA forms is quite similar. recA protein binds to B or Z DNA with a stoichiometry of 1 monomer/4 base pairs. The final protein filament exhibits a right-handed helical structure when either B or Z form DNAs are bound. There are only two evident differences: the kcat for ATP hydrolysis is reduced 3-4-fold when Z DNA is bound, and recA binding at equilibrium is less stable on Z DNA than on B DNA. At steady state, the binding favors B DNA in competition experiments. The results indicate that Z DNA binding by recA protein follows the same pathway as for recA binding to B DNA, but that the nucleation step is faster on the Z form helix.     

A Small Angle X-Ray Scattering Study of the Binding of Cyclosporin-A to Calmodulin

Small angle X-ray scattering (SAXS) was applied to the binding of the immunosuppressant drug cyclasporin-A to the protein calmodulin. Guinier analysis of the SAXS profiles yielded a radius of gyration, Rg, of 19.7 ± 0.3 Å for the native protein and 16.9 ± 0.3 Å for the drug/protein complex. Maximum entropy (maxent) methods of data analysis were used to calculate the distance distribution function, p(r). From this analysis, the R_g for the native protein is 20.9 ± 0.1 Å and that for the complex 16.7 ± 0.1 Å. The measured SAXS profiles and the derived p(r) for calmodulin agree with profiles calculated from the crystallographic structure of calmodulin. Major structural changes are induced in calmodulin on binding cyclosporin-A. A model consistent with the observed scattering profiles is an ellipsoid with major axes 55 and 36 Å. Molecular modeling of the calmodulin molecule suggests that bond rotation in the flexible α-helix linker region produces models consistent with the above observations.     

New Crystal Forms of the Motile Major Sperm Protein (MSP) ofAscaris suum

We have obtained two new crystal forms of theAscarismajor sperm protein (MSP) that mediates amoeboid cell motility in nematode sperm. We obtained crystals with C2 symmetry from bacterially expressed α-MSP witha= 216.5 Å,b= 38.6 Å,c= 32.5 Å, γ = 93.1° and also crystals with P2₁symmetry from native β-MSP witha= 63.1 Å,b= 91.7 Å,c= 72.5 Å, γ = 91.3°. A full native data set has been collected for each crystal form using synchrotron radiation. Both crystal forms diffract to 2 Å and are suitable for high-resolution structural investigation.     

recA protein filaments bind two molecules of single-stranded DNA with off rates regulated by nucleotide cofactor

To probe the role of nucleotide cofactor in the binding of single-stranded DNA to recA protein, we have developed a sedimentation assay using 5'-labeled 32P-poly(dT).recA.poly(dT) complexes sediment quantitatively when centrifuged at 100,000 x g for 45 min, whereas free poly(dT) remains in the supernatant. In the presence of ATP, between 6 and 7 bases cosediment per recA monomer; but when ADP is present or in the absence of added nucleotide cofactor, only 3-3.5 bases/recA monomer cosediment. In competition experiments in which recA.32P-poly(dT) complexes are incubated with unlabeled poly(dT), we again find 3-3.5 bases of labeled poly(dT) cosedimenting per recA monomer when no nucleotide cofactor is present. However, when the same experiment is performed with ATP, only half of the expected 6-7 bases of labeled poly(dT) remain bound to the DNA, demonstrating that half of the poly(dT) in the complex exchanges rapidly with free poly(dT), whereas the other half equilibrates slowly, like poly(dT) in the absence of nucleotide. The rate of exchange of the second more tightly bound poly(dT) is accelerated when ADP is present. Our observations are rationalized by a model in which each recA protein helical filament binds two strands of poly(dT) with a stoichiometry of 3-3.5 bases/recA monomer/strand.     

Activation of recA protein: the pitch of the helical complex with single-stranded DNA.

The complex of recA protein with single-stranded DNA in the presence of ATP is the active species in the three enzymatic activities of recA: the initiation of strand exchange, the hydrolysis of ATP and the cleavage of repressors. Here we find by cryo-electron microscopy of unstained and unfixed samples that the helical structure of the protein coat in this complex differs slightly but significantly from the structure in the complex with double-stranded DNA. We discuss how the larger pitch of the complex with single strands (100 +/- 2 A compared with 95 +/- 2 A with double strands) could contribute to its higher enzymatic activity.     

The distribution of Escherichia coli recA protein bound to duplex DNA with single-stranded ends

A short single-stranded tail on one end of an otherwise duplex DNA molecule enables recA protein, in the presence of ATP and MgCl2, to form a complex with the DNA which extends into the duplex portion of the molecule. Nuclease protection studies at a concentration of MgCl2 which permits homologous pairing showed that cleavage by restriction endonucleases at sites throughout the duplex region was inhibited, whereas digestion by DNase I was not affected. These results indicate that recA protein binds to the duplex portion of tailed DNA allowing access by DNase I to a random sample of the many sites at which it cleaves, but providing limited protection of the relatively rare restriction sites. Electron microscopy revealed that the recA nucleoprotein complex with duplex DNA is indeed a segmented or interrupted filament that, with time, extends further from the single-stranded tail into the duplex region. recA protein binding extended into the duplex region more rapidly for duplexes with 5' tails than for those with 3' tails. These observations show that recA protein translocates from a single-stranded region into duplex DNA in the form of a segmented filament by a mechanism that is not strongly polarized.     

Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA.

In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining. With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments. Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex. In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S. These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.     

Organized unidirectional waves of ATP hydrolysis within a RecA filament

The RecA protein forms nucleoprotein filaments on DNA, and individual monomers within the filaments hydrolyze ATP. Assembly and disassembly of filaments are both unidirectional, occurring on opposite filament ends, with disassembly requiring ATP hydrolysis. When filaments form on duplex DNA, RecA protein exhibits a functional state comparable to the state observed during active DNA strand exchange. RecA filament state was monitored with a coupled spectrophotometric assay for ATP hydrolysis, with changes fit to a mathematical model for filament disassembly. At 37 °C, monomers within the RecA-double-stranded DNA (dsDNA) filaments hydrolyze ATP with an observed k_cat of 20.8 ± 1.5 min⁻¹. Under the same conditions, the rate of end-dependent filament disassembly (k_off) is 123 ± 16 monomers per minute per filament end. This rate of disassembly requires a tight coupling of the ATP hydrolytic cycles of adjacent RecA monomers. The relationship of k_cat to k_off infers a filament state in which waves of ATP hydrolysis move unidirectionally through RecA filaments on dsDNA, with successive waves occurring at intervals of approximately six monomers. The waves move nearly synchronously, each one transiting from one monomer to the next every 0.5 s. The results reflect an organization of the ATPase activity that is unique in filamentous systems, and could be linked to a RecA motor function.     

Polymeric Structures and Dynamic Properties of the Bacterial Actin AlfA

AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 Å, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed. Here AlfA adopted a 16/7 helical symmetry, with a repeat of about 387 Å. Thin polymers consisting of several intertwining filaments also formed. Observed helical symmetries of AlfA filaments differed from those of other members of the actin family: F-actin, ParM, or MreB. Both ATP and guanosine 5′-triphosphate are able to promote rapid AlfA filament formation with almost equal efficiencies. The helical structure is only preserved under physiological salt concentrations and at a pH between 6.4 and 7.4, the physiological range of the cytoplasm of B. subtilis. Polymerization kinetics are extremely rapid and compatible with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation, making AlfA one of the most efficient polymerizing motors within the actin family. Phosphate release lags behind polymerization, and time-lapse total internal reflection fluorescence images of AlfA bundles are consistent with treadmilling rather than dynamic microtubule-like instability. High-pressure small angle X-ray scattering experiments reveal that the stability of AlfA filaments is intermediate between the stability of ParM and the stability of F-actin. These results emphasize that actin-like polymerizing machineries have diverged to produce a variety of filament geometries with diverse properties that are tailored for specific biological processes.