Interaction of the recA protein of Escherichia coli with single-stranded DNA

The interaction of recA protein with single-stranded (ss) phi X174 DNA has been examined by means of a nuclease protection assay. The stoichiometry of protection was found to be 1 recA monomer/approximately 4 nucleotides of ssDNA both in the absence of a nucleotide cofactor and in the presence of ATP. In contrast, in the presence of adenosine 5'-O-(thiotriphosphate) (ATP gamma S) the stoichiometry was 1 recA monomer/approximately 8 nucleotides. No protection was seen with ADP. In the absence of a nucleotide cofactor, the binding of recA protein to ssDNA was quite stable as judged by equilibration with a challenge DNA (t1/2 approximately 30 min). Addition of ATP stimulated this transfer (t1/2 approximately 3 min) as did ADP (t1/2 approximately 0.2 min). ATP gamma S greatly reduced the rate of equilibration (t1/2 greater than 12 h). Direct visualization of recA X ssDNA complexes at subsaturating recA protein concentrations using electron microscopy revealed individual ssDNA molecules partially covered with recA protein which were converted to highly condensed networks upon addition of ATP gamma S. These results have led to a general model for the interaction of recA protein with ssDNA.     

A partially deficient mutant, recA1730, that fails to form normal nucleoprotein filaments.

Summary The phenotype of the recA1730 mutant is highly dependent on the level of expression of the RecA1730 protein. If the recA1730 gene was expressed from its own promoter, the cells were deficient in recombination and SOS induction. In contrast, when the recA1730 gene was expressed under the control of recAo98, a constitutive operator that increased the RecA1730 concentration 20-fold, cells became proficient in recombination and SOS induction. Likewise, in crude extracts, fivefold more RecA1730 than RecAwt was required to produce full cleavage of LexA protein. The requirement for a high RecA1730 concentration for recombination and LexA cleavage suggests that the recA1730 defect alters a common reaction step. In fact, in vitro data show that the impaired assembly of RecA1730 protein on single-stranded DNA (ssDNA) can account for the mutant phenotype. Purified RecA1730 protein was assayed in vitro for ssDNA binding and ATPase activities. RecA1730, like RecAwt, retained ssDNA equally well on nitrocellulose filters; this activity was specifically inhibited by a monoclonal anti-RecA antibody. However, RecA1730 protein did not form complete filaments on ssDNA, as shown by two observations: (i) most of the protein did not elute with ssDNA during gel filtration; and (ii) binding of RecA1730 to ssDNA did not protect it from being digested by DNaseI. RecA1730 hydrolysed ATP in high salt but was defective in ssDNA-dependent ATP hydrolysis. These results strongly suggest that RecA1730 binds to ATP and ssDNA but does not form normal nucleoprotein filaments.Abbreviations RecAwt RecA wind-type protein - ssDNA singlestranded DNA - dsDNA dmble-stranded DNA     

Biochemical basis of the constitutive repressor cleavage activity of recA730 protein. A comparison to recA441 and recA803 proteins.

The recA730 mutation results in constitutive SOS and prophage induction. We examined biochemical properties of recA730 protein in an effort to explain the constitutive activity observed in recA730 strains. We find that recA730 protein is more proficient than the wild-type recA protein in the competition with single-stranded DNA binding protein (SSB protein) for single-stranded DNA (ssDNA) binding sites. Because an increased aptitude in the competition with SSB protein has been previously reported for recA441 protein and recA803 protein, we directly compared their in vitro activities with those of recA730 protein. At low magnesium ion concentration, both ATP hydrolysis and lexA protein cleavage experiments demonstrate that these recA proteins displace SSB protein from ssDNA in a manner consistent with their in vivo repressor cleavage activity, i.e. recA730 protein > recA441 protein > recA803 protein > recAwt protein. Additionally, a correlation exists between the proficiency of the recA proteins in SSB protein displacement and their rate of association with ssDNA. We propose that an increased rate of association with ssDNA allows recA730 protein to displace SSB protein from the ssDNA that occurs naturally in Escherichia coli and thereby to become activated for the repressor cleavage that leads to SOS induction. RecA441 protein is similarly activated for repressor cleavage; however, in this case, significant SSB protein displacement occurs only at elevated temperature. At physiological magnesium ion concentration, we argue that recA803 protein and wild-type recA protein do not displace sufficient SSB protein from ssDNA to constitutively induce the SOS response.     

Properties of the high-affinity single-stranded DNA binding state of the Escherichia coli recA protein

The properties of the high-affinity single-stranded DNA (ssDNA) binding state of Escherichia coli recA protein have been studied. We find that all of the nucleoside triphosphates that are hydrolyzed by recA protein induce a high-affinity ssDNA binding state. The effect of ATP binding to recA protein was partially separated from the ATP hydrolytic event by substituting calcium chloride for magnesium chloride in the binding buffer. Under these conditions, the rate of ATP hydrolysis is greatly inhibited. ATP increases the affinity of recA protein for ssDNA in a concentration-dependent manner in the presence of both calcium and magnesium chloride with apparent Kd values of 375 and 500 microM ATP, respectively. Under nonhydrolytic conditions, the molar ratio of ATP to ADP has an effect on the recA protein ssDNA binding affinity. Over an ATP/ADP molar ratio of 2-3, the affinity of recA protein for ssDNA shifts cooperatively from a low-to a high-affinity state.     

The physical and enzymatic properties of Escherichia coli recA protein display anion-specific inhibition.

The enzymatic activities of Escherichia coli recA protein are sensitive to ionic composition. Here we report that sodium glutamate (NaGlu) is much less inhibitory to the DNA strand exchange, DNA-dependent ATPase, and DNA binding activities of the recA protein than is NaCl. Both joint molecule formation and complete exchange of DNA strands occur (albeit at reduced rates) at NaGlu concentrations as high as 0.5 M whereas concentrations of NaCl greater than 0.2 M are sufficient for complete inhibition. The single-stranded DNA (ssDNA)-dependent ATPase activity is even less sensitive to inhibition by NaGlu; ATP hydrolysis stimulated by M13 ssDNA is unaffected by 0.5 M NaGlu and is further stimulated by E. coli ssDNA binding protein approximately 2-fold. Finally, NaGlu has essentially no effect on the stability of recA protein-epsilon M13 DNA complexes, with concentrations of NaGlu as high as 1.5 M failing to dissociate the complexes. Surprisingly, NaGlu also has little effect on the concentration of NaCl required to disrupt the recA protein-epsilon M13 DNA complex, demonstrating that destabilization is dependent on both the concentration and type of anionic rather than cationic species. Quantitative analysis of DNA binding isotherms establishes that the intrinsic binding affinity of recA protein is affected by the anionic species present and that the cooperativity parameter is relatively unaffected. Consequently, the sensitivity of recA protein-ssDNA complexes to disruption by NaCl does not result from the competitive effects associated with cation displacement from the ssDNA upon protein binding but rather results from anion displacement upon complex formation. The magnitude of this anion-specific effect on ssDNA binding is large relative to that of other nucleic acid binding proteins.     

Exchange of recA protein between adjacent recA protein-single-stranded DNA complexes

We have examined the exchange of recA protein between stable complexes formed with single-stranded DNA (ssDNA) and (a) other complexes and (b) a pool of free recA protein. We have also examined the relationship of ATP hydrolysis to these exchange reactions. Exchange was observed between two different recA X ssDNA complexes in the presence of ATP. Complete equilibration between two sets of complexes occurred with a t1/2 of 3-7 min under a set of conditions previously found to be optimal for recA protein-promoted DNA strand exchange. Approximately 200 ATPs were hydrolyzed for every detected migration of a recA monomer from one complex to another. This exchange occurred primarily between adjacent complexes, however. Little or no exchange was observed between recA X ssDNA complexes and the free recA protein pool, even after several hundred molecules of ATP had been hydrolyzed for every recA monomer present. ATP hydrolysis is not coupled to complete dissociation or association of recA protein from or with recA X ssDNA complexes under these conditions.     

Active nucleoprotein filaments of single-stranded binding protein and recA protein on single-stranded DNA have a regular repeating structure.

When E. coli single-stranded DNA binding protein (SSB) coats single-stranded DNA (ssDNA) in the presence of 1 mM MgCl2 it inhibits the subsequent binding of recA protein, whereas SSB binding to ssDNA in 12 mM MgCl2 promotes the binding of recA protein. These two conditions correspond respectively to those which produce 'smooth' and 'beaded' forms of ssDNA-SSB filaments. By gel filtration and immunoprecipitation we observed active nucleoprotein filaments of recA protein and SSB on ssDNA that contained on average 1 monomer of recA protein per 4 nucleotides and 1 monomer of SSB per 20-22 nucleotides. Filaments in such a mixture, when digested with micrococcal nuclease produced a regular repeating pattern, approximately every 70-80 nucleotides, that differed from the pattern observed when only recA protein was bound to the ssDNA. We conclude that the beaded ssDNA-SSB nucleoprotein filament readily binds recA protein and forms an intermediate that is active in the formation of joint molecules and can retain substantially all of the SSB that was originally bound.     

Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage.

Evidence is presented that recA functions which promote the SOS functions of mutagenesis, LexA protein proteolysis, and lambda cI repressor proteolysis are each genetically separable from the others. This separation was observed in recombination-proficient recA mutants and rec+ (F' recA56) heterodiploids. recA430, recA433, and recA435 mutants and recA+ (F' recA56) heterodiploids were inducible for only one or two of the three functions and defective for mutagenesis. recA80 and recA432 mutants were constitutively activated for two of the three functions in that these mutants did not have to be induced to express the functions. We propose that binding of RecA protein to damaged DNA and subsequent interaction with small inducer molecules gives rise to conformational changes in RecA protein. These changes promote surface-surface interactions with other target proteins, such as cI and LexA proteins. By this model, the recA mutants are likely to have incorrect amino acids substituted as sites in the RecA protein structure which affect surface regions required for protein-protein interactions. The constitutively activated mutants could likewise insert altered amino acids at sites in RecA which are involved in the activation of RecA protein by binding small molecules or polynucleotides which metabolically regulate RecA protein.     

Stabilization of recA protein-ssDNA complexes by the single-stranded DNA binding protein of Escherichia coli

In vitro recombination reactions promoted by the recA protein of Escherichia coli are enhanced by the single-stranded DNA binding protein (SSB). SSB affects the assembly of the filamentous complexes between recA protein and ssDNA that are the active form of the recA protein. Here, we present evidence that SSB plays a complex role in maintaining the stability and activity of recA-ssDNA filaments. Results of ATPase, nuclease protection, and DNA strand exchange assays suggest that the continuous presence of SSB is required to maintain the stability of recA-ssDNA complexes under reaction conditions that support their recombination activity. We also report data that indicate that there is a functional distinction between the species of SSB present at 10 mM magnesium chloride, which enhances recA-ssDNA binding, and a species present at 1 mM magnesium chloride, which displaces recA protein from ssDNA. These results are discussed in the context of current models of SSB conformation and of SSB action in recombination activities of the recA protein.     

Distances between functional sites of the Ca2+ + Mg2(+)-ATPase from sarcoplasmic reticulum using Co2+ as a spectroscopic ruler

Cobalt ion inhibits the Ca2+ + Mg2(+)-ATPase activity of sealed sarcoplasmic reticulum vesicles, of solubilized membranes and of the purified enzyme. To use Co2+ appropriately as a spectroscopic ruler to map functional sites of the Ca2+ + Mg2(+)-ATPase, we have carried out studies to obtain the kinetic parameters needed to define the experimental conditions to conduct the fluorimetric studies. 1. The apparent K0.5 values of inhibition of this ATPase are 1.4 mM, 4.8 mM and 9.5 mM total Co2+ at pH 8.0, 7.0 and 6.0, respectively. The inhibition by Co2+ is likely to be due to free Co2+ binding to the enzyme. Millimolar Ca2+ can fully reverse this inhibition, and also reverses the quenching of the fluorescence of fluorescein-labeled sarcoplasmic reticulum membranes due to Co2+ binding to the Ca2+ + Mg2(+)-ATPase. Therefore, we conclude that Co2+ interacts with Ca2+ binding sites. 2. Co2+.ATP can be used as a substrate by this enzyme with Vmax of 2.4 +/- 0.2 mumol ATP hydrolyzed min-1 (mg protein)-1 at 20-22 degrees C and pH 8.0, and with a K0.5 of 0.4-0.5 mM. 3. Co2+ partially quenches, about 10 +/- 2%, the fluorescence of fluorescein-labeled sarcoplasmic reticulum Ca2+ + Mg2(+)-ATPase upon binding to this enzyme at pH 8.0. From the fluorescence data we have estimated an average distance between Co2+ and fluorescein in the ATPase of 1.1-1.8 nm or 1.3-2.1 nm for one or two equidistant Co2+ binding sites, respectively. 4. Co2+.ATP quenches about 20-25% of the fluorescence of fluorescein-labeled Ca2+ + Mg2(+)-ATPase, from which we obtain a distance of 1.1-1.9 nm between Co2+ and fluorescein located at neighbouring catalytic sites.     

Biochemical properties of the Escherichia coli recA430 protein. Analysis of a mutation that affects the interaction of the ATP-recA protein complex with single-stranded DNA

The biochemical properties of the recA430 protein have been examined and compared to those of wild-type recA protein. We find that, while the recA430 protein possesses ssDNA-dependent rATP activity, this activity is inhibited by the Escherichia coli single-stranded DNA binding protein (SSB protein) under many conditions that enhance wild-type recA protein rATPase hydrolysis. Stimulation of rATPase activity by SSB protein is observed only at high concentrations of both rATP (greater than 1 mM) and recA430 protein (greater than 5 microM). In contrast, stimulation of ssDNA-dependent dATPase activity by SSB protein is less sensitive to protein and nucleotide concentration. Consistent with the nucleotide hydrolysis data, recA430 protein can carry out DNA strand exchange in the presence of either rATP or dATP. However, in the presence of rATP, both the rate and the extent of DNA strand exchange by recA430 protein are greatly reduced compared to wild-type recA protein and are sensitive to recA430 protein concentration. This reduction is presumably due to the inability of recA430 protein to compete with SSB protein for ssDNA binding sites under these conditions. The cleavage of lexA repressor protein by recA430 protein is also sensitive to the nucleotide cofactor present and is completely inhibited by SSB protein when rATP is the cofactor but not when dATP is used. Finally, the steady-state affinity and the rate of association of the recA430 protein-ssDNA complex are reduced, suggesting that the mutation affects the interaction of the ATP-bound form of recA protein with ssDNA. This alteration is the likely molecular defect responsible for inhibition of recA430 protein rATP-dependent function by SSB protein. The biochemical properties observed in the presence of dATP and SSB protein, i.e. the reduced levels of both DNA strand exchange activity and cleavage of lexA repressor protein, are consistent with the phenotypic behavior of recA430 mutations.     

Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes.

1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.     

Enhancement of Escherichia coli RecA protein enzymatic function by dATP

The Escherichia coli recA protein has been shown to hydrolyze several nucleoside triphosphates in the presence of ssDNA. The substitution of dATP for rATP has significant effects on various recA protein biochemical properties. In the presence of dATP, recA protein can invade more secondary structure in native ssDNA than it can in the presence of rATP. The dATP-recA protein complex can compete more effectively with the E. coli ssDNA binding protein (SSB) for ssDNA binding sites compared with the rATP-recA protein complex. Finally, the rate of dATP hydrolysis stimulated by dsDNA is greater than the rate of rATP hydrolysis. These effects, in turn, are observed as alterations in the recA protein catalyzed DNA strand exchange reaction. In the absence of SSB protein, the rate of joint molecule and product formation in the DNA strand exchange reaction is greater in the presence of dATP than in the presence of rATP. The rate of product formation in the dATP-dependent reaction is also faster than the rATP-dependent reaction when SSB protein is added to the ssDNA before recA protein; the rate of rATP-dependent product formation is inhibited 10-fold under these conditions. This nucleotide, dATP, was previously shown to induce an apparent affinity of recA protein for ssDNA which is higher than any other NTP. These results suggest that the observed enhancement of enzymatic activity may be related to the steady-state properties of the high-affinity ssDNA binding state of recA protein. In addition, the data suggest that recA protein functions in NTP hydrolysis as a dimer of protein filaments and that the binding of ssDNA to only one of the recA filaments is sufficient to activate all recA protein molecules in the dimeric filament. The implications of this finding to the enzymatic function of recA protein are discussed.     

Activation of protease-constitutive recA proteins of Escherichia coli by rRNA and tRNA.

The RecA proteins of the unusually strong protease-constitutive mutants recA1202 and recA1211 can use RNA in addition to single-stranded DNA (ssDNA) as a cofactor in the cleavage of the LexA repressor in vitro. In the presence of rRNA or tRNA, the effectiveness of these proteins decreased in the order RecA1202 greater than RecA1211 much greater than RecA+, which is also the order of their in vivo constitutive protease activities. The effectiveness of rRNA was comparable to that of ssDNA in the cleavage of the LexA repressor by either mutant protease. Although all the common nucleoside triphosphates can act as positive effectors for LexA cleavage by the two mutant proteins in the presence of ssDNA (W. B. Wang, M. Sassanfar, I. Tessman, J. W. Roberts, and E. S. Tessman, J. Bacteriol. 170:4816-4822, 1988), only dATP, ATP, and ATP-gamma-S were effective in the presence of RNA. Our results explain more fully why certain recA mutants have high constitutive protease activities in vivo.     

Increased permeability and subsequent resealing of the host cell membrane early after infection of Escherichia coli with bacteriophage T1.

The addition of T1 to cells growing at 37 degrees C in a minimal medium at 0.4 mM Mg2+ rapidly induced an irreversible loss of K+ and Mg2+ and uptake of Na+ by the cells. Both the ATP pool of the cells and the transmembrane proton motive force were reduced. These cells did not lyse from within, since viral DNA replication and the maturation of the 36,000-molecular-weight phage head protein were inhibited. By contrast, cells lysed when infected at 5.4 mM Mg2+. In these cells, T1 initially induced K+ efflux and Na+ influx and lowered the cytoplasmic ATP concentration. After a few minutes, the cation gradients and ATP pool were restored to levels close to that of control cells. At 5.4 mM Mg2+, the shutoff of host protein synthesis was delayed and coincided with the restoration of the ATP pool. In an ATP synthase-negative mutant, infection with T1 did not affect the cytoplasmic ATP concentration but inhibited host protein synthesis with the same rate as it did in wild-type cells.     

Homology-dependent changes in adenosine 5'-triphosphate hydrolysis during recA protein promoted DNA strand exchange: evidence for long paranemic complexes

As a first step in DNA strand exchange, recA protein forms a filamentous complex on single-stranded DNA (ssDNA), which contains stoichiometric (one recA monomer per four nucleotides) amounts of recA protein. recA protein monomers within this complex hydrolyze ATP with a turnover number of 25 min-1. Upon introduction of linear homologous duplex DNA to initiate strand exchange, this rate of ATP hydrolysis drops by 33%. The decrease in rate is complete in less than 2 min, and the rate of ATP hydrolysis then remains constant during and subsequent to the strand exchange reaction. This drop is completely dependent upon homology in the duplex DNA. In addition, the magnitude of the drop is linearly dependent upon the length of the homologous region in the linear duplex DNA. Linear DNA substrates in which pairing is topologically restricted to a paranemic joint also follow this relationship. Taken together, these properties imply that all of the available homology in the incoming duplex DNA is detected very early in the DNA strand exchange reaction, with the linear duplex DNA paired paranemically with the homologous ssDNA in the complex throughout its length. The results indicate that paranemic joints can extend over thousands of base pairs. We note elsewhere [Pugh, B. F., & Cox, M. M. (1987b) J. Biol. Chem. 262, 1337-1343] that this duplex acquires resistance to digestion by DNase with a much slower time course (30 min), which parallels the progress of strand exchange. Together these results imply that the duplex DNA is paired with the ssDNA but remains outside the nucleoprotein filament. Finally, the results also support the notion that ATP hydrolysis occurs throughout the recA nucleoprotein filament.     

Enhancement of recA protein-promoted DNA strand exchange activity by volume-occupying agents.

To investigate the in vivo effects of macromolecular crowding we examined the effect of inert macromolecules such as polyvinyl alcohol and polyethylene glycol on the in vitro activity of recA protein. The addition of either of these volume-occupying agents enables recA protein to promote homologous pairing and exchange of DNA strands at an otherwise nonpermissive magnesium ion concentration. In the presence of these macromolecules, both the rate of recA protein association with single-stranded DNA (ssDNA) and the steady-state affinity of recA protein for ssDNA are increased. Consequently, the ability of recA protein to compete with ssDNA-binding protein (SSB protein) is enhanced, and the inhibitory effects of SSB protein on the formation of recA protein-ssDNA presynaptic complexes are eliminated. Because the ability of recA protein to bind to ssDNA-containing secondary structures is also enhanced in volume-occupied solution, joint molecule formation is not greatly reduced when SSB protein is omitted from the reaction. Thus, increased recA protein interactions with ssDNA contribute to enhanced presynaptic complex formation. In addition, polyvinyl alcohol and polyethylene glycol must also affect another property of recA protein, i.e. self-association, which is required for synapsis and DNA strand exchange. Our examination of DNA strand exchange in the presence of volume-occupying agents helps to reconcile the requirement for elevated magnesium ion concentrations in recA protein-promoted recombination reactions in vitro, with a presumably low magnesium ion concentration in vivo.     

Modulating action of barium and other ions on the sensory activity of frog labyrinth

The effect of Ba2+, TEA, 4-AP and CoCl2 on the EPSP and spike discharges recorded from single fibres of the posterior nerve in the isolated frog labyrinth has been investigated. In Ca-free solution Ba2+ preserved, at low concentration (0.3 mM), the resting activity and at higher levels (up to 6 mM) it resulted in a pronounced facilitation of the EPSP and spike discharges. Facilitation increased on increasing Ba2+ concentration up to 4-5 mM and it was more evident in those units exhibiting a low resting spike firing. The effect of Ba2+ (1 mM) was completely antagonized by 10 mM Ca2+ X CoCl2 (3 mM) suppressed the resting rate at the normal external Ca2+ concentration; the Co2+ block was partially relieved by 1.8 mM Ba2+ X TEA (20 mM) evoked a clear-cut increase in the EPSP and spike discharges which, however, was less consistent than that produced by Ba2+. By comparing the effect of TEA on the spike frequency with that obtained at different Ba2+ levels, the Ba2+ capacity to carry the Ca2+ current was dissected. Such an effect is dose-dependent and it is more evident in low-frequency units. Conversely, 4-AP did not affect the resting discharge frequency. These results indicate that either the Ca2+ or the Ba2+ current sustain the transmitter release at the cyto-neural junction. The effect of TEA suggests that the Ca2+-dependent K+ current may play an important role in supporting the neurosecretory process by controlling the membrane potential of the hair cells.     

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.     

Hormone-specific combinations of isoforms of adenylyl cyclase and phosphodiesterase in the rat liver

Since many isoforms of adenylyl cyclase and adenosine 3', 5'-monophosphate (cAMP) phosphodiesterase have been cloned, it is likely that receptors of each hormone have a specific combination of these isoforms. Types I, III and VIII adenylyl cyclases are reported to be stimulated by Ca(2+)-calmodulin, type I phosphodiesterase by Ca(2+)-calmodulin, but types IV and VII (cAMP-specific) phosphodiesterases by Co2+. In the present study, we examined different effects of Ca2+ and Co2+ on hormone-induced cAMP response in the isolated perfused rat liver.The removal of Ca2+ from the perfusion medium (0 mM CaCl(2 ) + 0.5 mM EGTA) did not affect glucagon (0.1 nM)-responsive cAMP but reduced secretin (1 nM)-, vasoactive intestinal polypeptide (VIP, 1-10 nM)- and forskolin (1 microM)-responsive cAMP considerably. The addition of 1 mM CoCl2 reduced glucagon- and secretin-responsive cAMP considerably, forskolin-responsive cAMP partly, did not affect 1 nM VIP-responsive cAMP, but enhanced 10 nM VIP-responsive cAMP. Forskolin- and VIP-responsive cAMP was greater in the combination (0 mM CaCl(2) + 0.5 mM EGTA + 3 mM CoCl2) than in the Ca(2+)-free perfusion alone.These results suggest that secretin, VIP1 and VIP2 receptors are linked to Ca(2+)-calmodulin-sensitive adenylyl cyclase; glucagon receptor to Ca(2+)-calmodulin-insensitive adenylyl cyclase; VIP1 receptor to Ca(2+)-calmodulin-dependent phosphodiesterase; glucagon, secretin and VIP2 receptors to cAMP-specific phosphodiesterase, respectively, in the rat liver.