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.     

Plaque color method for rapid isolation of novel recA mutants of Escherichia coli K-12: new classes of protease-constitutive recA mutants

As a prerequisite to mutational analysis of functional sites on the RecA protein of Escherichia coli, a method was developed for rapid isolation of recA mutants with altered RecA protease function. The method involves plating mutagenized lambda recA+ cI ind on strains deleted for recA and containing, as indicators of RecA protease activity, Mu d(Ap lac) fusions in RecA-inducible genes. The lambda recA phages were recognized by their altered plaque colors, and the RecA protease activity of the lambda recA mutant lysogens was measured by expression of beta-galactosidase from dinD::lac. One class of recA mutants had constitutive protease activity and was designated Prtc; in these cells the RecA protein was always in the protease form without the usual need for DNA damage to activate it. Some Prtc mutants were recombinase negative and were designated Prtc Rec-. Another class of 65 recA mutants isolated as being protease defective were all also recombinase defective. Unlike the original temperature-dependent Prtc Rec+ mutant (recA441), the new Prtc Rec+ mutants showed constitutive protease activity at any growth temperature, with some having considerably greater activity than the recA441 strain. Study of these strong Prtc Rec+ mutants revealed a new SOS phenomenon, increased permeability to drugs. Use of this new SOS phenomenon as an index of protease strength clearly distinguished 5 Prtc mutants as the strongest among 150. These five strongest Prtc mutants showed the greatest increase in spontaneous mutation frequency and were not inhibited by cytidine plus guanosine, which inhibited the constitutive protease activity of the recA441 strain and of all the other new Prtc mutants. Strong Prtc Rec+ mutants were more UV resistant than recA+ strains and showed indications of having RecA proteins whose specific activity of recombinase function was higher than that of wild-type RecA. A Prt+ Rec- mutant with an anomalous response to effectors is described.     

Activation of a latent RNAase from yeast by nucleoside triphosphates

A latent RNAase activity stimulated by nucleoside triphosphates has been isolated from a yeast chromatin extract, by filtration on Sepharose 6B and hydroxyapatite chromatography. The RNAase was separated from a thermolabile proteic inhibitor on phosphocellulose. When separated from the inhibitor, the RNAase hydrolyses RNA to 5′-mononucleotides. Its activity is retained in the presence of EDTA, and 50% inhibited by 1 mM ATP or CTP. The RNAase is inhibited by the thermolabile component only in the presence of divalent cations. The activity is recovered upon addition of 0.01 mM ATP to the mixture. The K_m for ATP is 10 μM. ATP can be replaced by other ribo- or deoxyribonucleoside triphosphates with varying efficiency but not by ADP, AMP or cAMP. These results suggest multiple interactions between the RNAase, a regulatory component, divalent cations and nucleoside triphosphates.     

Accumulation of pyrophosphate correlates with the increased level of nucleoside triphosphates in Escherichia coli

We measured the concentration of nucleoside triphosphates and inorganic pyrophosphate in Escherichia coli in conditions where nucleotide synthesis or nucleic acid synthesis was inhibited. The inhibitors that brought about an accumulation of some of the four ribonucleoside triphosphates also increased the pyrophosphate level. In a pyrimidine auxotrophic strain uracil starvation led to simultaneous accumulation of ATP and pyrophosphate, and they both rapidly returned to normal level when starvation was relieved. These results indicate the possible involvement of pyrophosphate in the reactions leading to the accumulation of nucleoside triphosphates.     

Activity of E. coli ClpA bound by nucleoside diphosphates and triphosphates

The Escherichia coli ClpA protein is a molecular chaperone that binds and translocates protein substrates into the proteolytic cavity of the tetradecameric serine protease ClpP. In the absence of ClpP, ClpA can remodel protein complexes. In order for ClpA to bind protein substrates targeted for removal or remodeling, ClpA requires nucleoside triphosphate binding to first assemble into a hexamer. Here we report the assembly properties of ClpA in the presence of the nucleoside diphosphates and triphosphates ADP, adenosine 5′-[γ-thio]triphosphate, adenosine 5′-(β,γ-imido)triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and adenosine diphosphate beryllium fluoride. In addition to examining the assembly of ClpA in the presence of various nucleotides and nucleotide analogues, we have also correlated the assembly state of ClpA in the presence of these nucleotides with both polypeptide binding activity and enzymatic activity, specifically ClpA-catalyzed polypeptide translocation. Here we show that all of the selected nucleotides, including ADP, promote the assembly of ClpA. However, only adenosine 5′-[γ-thio]triphosphate and adenosine 5′-(β,γ-imido)triphosphate promote the formation of an oligomer of ClpA that is active in polypeptide binding and translocation. These results suggest that the presence of γ phosphate may serve to switch ClpA into a conformational state with high peptide binding activity, whereas affinity is severely attenuated when ADP is bound.     

Mutagenesis by proximity to the recA gene of Escherichia coli

Escherichia coli recA (Prtc) strains, which produce protease constitutive RecA proteins in the absence of DNA-damaging treatments, display an increased frequency of spontaneous mutations. These mutations occurred preferentially in the neighborhood of the recA gene. This cis-like mutagenic effect was observed in the recA, rexAB, phoE and bio genes. The localized mutagenesis can be explained by the ease with which RecA(Prtc) proteins are activated to the protease state, which implies that there should be a relatively high concentration of activated RecA protein near the recA gene, where the protein is synthesized. The unusually high frequency of mutation in the recA gene is a novel example of an overactive gene preferentially turning itself down by mutation.     

Formation of heteroduplex DNA promoted by the combined activities of Escherichia coli recA and recBCD proteins

We have established an in vitro reaction in which heteroduplex DNA formation is dependent on the concerted actions of recA and recBCD proteins, the major components of the recBCD pathway of genetic recombination in vivo. We find that heteroduplex DNA formation requires three distinct enzymatic functions: first, the helicase activity of recBCD enzyme initiates heteroduplex DNA formation by unwinding the linear double-stranded DNA molecule to transiently form single-stranded DNA (ssDNA); second, recA protein traps this ssDNA before it reanneals; third, recA protein catalyzes the pairing of this ssDNA molecule with another homologous ssDNA molecule, followed by the renaturation of these molecules to form heteroduplex DNA. The first two functions should be important to all in vitro reactions involving recA and recBCD proteins, whereas the third will be specific to the DNA substrates used. The rate-limiting step of heteroduplex DNA formation is the trapping by recA protein of the ssDNA produced by recBCD enzyme. A model for this reaction is described.     

Left-handed Z-DNA binding by the recA protein of Escherichia coli

recA binding to left-handed Z-DNA was measured using nitrocellulose filter binding assays with four DNA polymers with defined nucleotide sequences and four recombinant plasmids. Two to 7-fold preferential binding of recA to Z-DNA polymers was observed. Left-handed Z-DNA polymer binding by recA required ATP or its nonhydrolyzable analog, ATP(gamma S), while ADP inhibited binding. Complex formation with both B- and Z-forms was influenced by polymer length; recA bound longer DNAs better. recA binding to recombinant plasmids containing supercoil-stabilized Z-DNA was essentially similar to that found for the control vector; thus, no preferential binding of recA to the Z-form was observed. Comparative experiments with the rec1 protein of Ustilago maydis and the Escherichia coli recA protein were performed. In our hands, recA and rec1 have a similar capacity for binding left-handed Z-DNA polymers and for binding recombinant plasmids containing B- and/or Z-regions. recA contains a left-handed Z-DNA-stimulated ATPase activity. This activity differs from the right-handed B-DNA-stimulated activity since it is less sensitive to increasing pH. The kinetics of ATP hydrolysis in B-DNA/Z-DNA mixing experiments showed that the turnover of the Z-DNA recA complex was slower than for B-DNA suggesting that left-handed Z-DNA is more stably bound by recA. Our results are consistent with the postulate that left-handed Z-DNA is involved in genetic recombination.     

Ribosomal abnormality in recA mutants of Escherichia coli.

Summary The tif-1 mutation has been shown to affect protein synthesis in vitro by increasing translational ambiguity (Ephrati-Elizur, Luther-Davies and Hayes, 1976). It is demonstrated here that some recA mutations confer similar abnormality. By comparing suitable combinations of ribosomes and soluble proteins from recA ⁺ and recA cells the defect is shown to be associated with ribosomes. The recA mutation, which suppresses most phenotypic characteristics of the tif-1 mutation (Castellazzi, George and Buttin, 1972(b)) does not suppress the ribosomal abnormality. Sience the closely linked tif-1 and recA mutations lead to the expression of a common property they may be in the same gene.     

Escherichia coli protein X is the recA gene product.

Escherichia coli protein X is known to be made in large amounts following DNA damage or inhibition of DNA replication. We have shown that it is identical to the recA gene product by partial proteolytic digestion of the radiochemically pure proteins and analysis by electrophoresis on polyacrylamide-sodium dodecyl sulfate gels.     

Function of nucleoside triphosphate and polynucleotide in Escherichia coli recA protein-directed cleavage of phage lambda repressor

Escherichia coli recA protein catalyzes a specific proteolytic cleavage of repressors in vitro when it is activated by interaction with a single-stranded polynucleotide and nucleoside triphosphate. The ATP analogue adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) satisfies the NTP requirement. We show here that despite its activity in repressor cleavage, ATP gamma S is hydrolyzed at a negligible rate by the recA protein DNA-dependent nucleoside triphosphatase activity. In the presence of DNA, ATP gamma S binds tightly to recA protein in a complex that can be detected because it is trapped by a nitrocellulose filter. One ATP gamma S molecule is bound per recA monomer. These results suggest that a ternary complex of recA protein, DNA, and nucleoside triphosphate is the species active in repressor cleavage. The activation of recA protein by small, defined oligonucleotides in place of DNA is described and characterized.     

Convenient construction of recA deletion derivatives of Escherichia coli.

Two Escherichia coli K-12 Hfr strains have been constructed which transfer a recA deletion, which is highly linked to a Tn10 insertion conferring tetracycline resistance, early during conjugation. These strains transfer the recA deletion in opposite directions with different origins of transfer, allowing for preservation of desirable recipient strain markers either clockwise or counterclockwise of recA.     

Location of functional regions of the Escherichia coli RecA protein by DNA sequence analysis of RecA protease-constitutive mutants.

In previous work (E. S. Tessman and P. K. Peterson, J. Bacteriol. 163:677-687 and 688-695, 1985), we isolated many novel protease-constitutive (Prtc) recA mutants, i.e., mutants in which the RecA protein was always in the protease state without the usual need for DNA damage to activate it. Most Prtc mutants were recombinase positive and were designated Prtc Rec+; only a few Prtc mutants were recombinase negative, and those were designated Prtc Rec-. We report changes in DNA sequence of the recA gene for several of these mutants. The mutational changes clustered at three regions on the linear RecA polypeptide. Region 1 includes amino acid residues 25 through 39, region 2 includes amino acid residues 157 through 184, and region 3 includes amino acid residues 298 through 301. The in vivo response of these Prtc mutants to different effectors suggests that the RecA effector-binding sites have been altered. In particular we propose that the mutations may define single-stranded DNA- and nucleoside triphosphate-binding domains of RecA, that polypeptide regions 1 and 3 comprise part of the single-stranded DNA-binding domain, and that polypeptide regions 2 and 3 comprise part of the nucleoside triphosphate-binding domain. The overlapping of single-stranded DNA- and nucleoside triphosphate-binding domains in region 3 can explain previously known complex allosteric effects. Each of four Prtc Rec- mutants sequenced was found to contain a single amino acid change, showing that the change of just one amino acid can affect both the protease and recombinase activities and indicating that the functional domains for these two activities of RecA overlap. A recA promoter-down mutation was isolated by its ability to suppress the RecA protease activity of one of our strong Prtc mutants.