Roles of RecA protein in spontaneous mutagenesis in Escherichia coli

To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations. In a recA null mutant, no recombination event was observed. These data suggest that the blockage of DNA replication, probably caused by spontaneous DNA lesions, occurs often in normally growing E. coli cells and is mainly processed by cellular functions requiring the RecA protein. However, the recA mutant strain showed elevated frequencies of single-base frameshifts and large deletions, implying a novel mutator action of this strain. A similar mutator action of the recA mutant was also observed with a plasmid-based rpsL mutation assay. Therefore, if the recombinogenic problems in DNA replication are not properly processed by the RecA function, these would be a potential source for mutagenesis leading to single-base frameshift and large deletion in E. coli. Furthermore, the single-base frameshifts induced in the recA-deficient cells appeared to be efficiently suppressed by the mutS-dependent mismatch repair system. Thus, it seems likely that the single-base frameshifts are derived from slippage errors that are not directly caused by DNA lesions but made indirectly during some kind of error-prone DNA synthesis in the recA mutant cells.     

RecA protein of Escherichia coli and chromosome partitioning

Escherichia coli cells deficient in RecA protein frequently contain an abnormal number of chromosomes after completion of ongoing rounds of DNA replication. This suggests that RecA protein may be required for correct timing of initiation of DNA replication; however, we show here that initiation of DNA replication is properly timed in recA mutants. We also find that more than 10% of recA mutant cells contain no DNA. These anucleate cells appear to arise from partitioning of all the DNA into one daughter cell and no DNA into the other daughter cell. Based on these and previously published results, we propose that RecA protein is required for equal partitioning of chromosomes into the two daughter cells.     

Relationship between the functional regions of the RecA protein and ATP hydrolysis in UV-irradiated Escherichia coli cells.

The time course of the intracellular ATP concentration in several UV-irradiated RecA protease constitutive (Cptc) mutants of E. coli has been studied. All Cptc mutants harboring a mutation in region 3 of the RecA protein (including amino acid residues 298-301) increased ATP after UV damage but without any subsequent decrease. Nevertheless, these mutants induced the SOS response after UV irradiation. Likewise, truncated RecA proteins lacking region 3 are also unable to carry out massive ATP hydrolysis in UV-irradiated cells. On the other hand, mutants in region 1 (including amino acids 25-39) or 2 (amino acids 157-184) of the RecA protein showed an increase in ATP concentration during the first 20 min following UV irradiation, which dropped afterwards to the basal level. All these data indicate that region 3 of the RecA protein must be involved in the ATP hydrolysis process. Furthermore, a relationship between the quantity of the UV-mediated ATP produced and the strength of the different RecA Cptc mutants has also been found. Accordingly, both lexA71::Tn5 and null lexA mutants of E. coli only show a cellular ATP increase after UV irradiation when containing a multicopy plasmid carrying either a wild-type lexA or a lexA (Ind-) gene.     

New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis.

To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication. By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations. A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination. The mutation rendered the host not mutable by UV, even in a lexA(Def) background. Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD. Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein. Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination. The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination. Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage. Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates. The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+. With respect to other RecA functions, recA1730 was recessive to recA+. This demonstrates that RecA protein has an additional role in mutagenesis beside mediating the cleavage of LexA and UmuD proteins.     

A yeast protein analogous to Escherichia coli RecA protein whose cellular level is enhanced after UV irradiation

Summary In Saccharomyces cerevisiae, a protein was recognized by polyclonal antibodies raised against homogeneous Escherichia coli K12 RecA protein. The cellular level of the yeast protein called RecAsc (molecular weight 44 kDa, pI 6.3), was transiently enhanced after UV irradiation. Protease inhibitors were required to minimize degradation of the RecAsc protein during cell lysis. The RecAsc protein exhibited similar basal levels and similar kinetics of increase after UV irradiation in DNA-repair proficient (RAD ⁺) strains carrying mitochondrial DNA or not (rho ⁰). This was also true for the following DNA-repair deficient (rad ^-) strains: rad2-6 rad6-1 rad52-1, a triple mutant blocked in three major repair pathways; rad6-, a mutant containing an integrative deletion in a gene playing a central role in mutagenesis; pso2-1, a mutant that exhibits a reduced rate of mutagenesis and recombination after exposure to DNA cross-linking agents.     

The Escherichia coli DinD protein modulates RecA activity by inhibiting postsynaptic RecA filaments

Escherichia coli dinD is an SOS gene up-regulated in response to DNA damage. We find that the purified DinD protein is a novel inhibitor of RecA-mediated DNA strand exchange activities. Most modulators of RecA protein activity act by controlling the amount of RecA protein bound to single-stranded DNA by affecting either the loading of RecA protein onto DNA or the disassembly of RecA nucleoprotein filaments bound to single-stranded DNA. The DinD protein, however, acts postsynaptically to inhibit RecA during an on-going DNA strand exchange, likely through the disassembly of RecA filaments. DinD protein does not affect RecA single-stranded DNA filaments but efficiently disassembles RecA when bound to two or more DNA strands, effectively halting RecA-mediated branch migration. By utilizing a nonspecific duplex DNA-binding protein, YebG, we show that the DinD effect is not simply due to duplex DNA sequestration. We present a model suggesting that the negative effects of DinD protein are targeted to a specific conformational state of the RecA protein and discuss the potential role of DinD protein in the regulation of recombinational DNA repair.     

PsiB polypeptide prevents activation of RecA protein in Escherichia coli

Summary We further characterize a novel plasmid function preventing SOS induction called Psi (Plasmid SOS Inhibition). We show that Psi function is expressed by psiB, a gene located at coordinate 54.9 of plasmid R6-5 and near oriT, the origin of conjugal transfer. Deletions and amber mutations of the psiB gene permitted us to demonstrate that PsiB polypeptide (apparent molecular weight, 12 kDa) is responsible for Psi function. PsiB protein prevents recA730-promoted mutagenesis and intra-chromosomal recombination but not recombination following conjugation. Overproduction of PsiB protein sensitizes the host cell to UV irradiation. We propose that PsiB polypeptide has an anti-SOS action by inhibiting activation of RecA protein, thus preventing the occurrence of LexA-controlled functions.     

Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein

The RecA protein of Escherichia coli plays important roles in homologous recombination, recombinational DNA repair, and SOS induction. Because its functions are conserved among the phylogenetic kingdoms, RecA investigations have provided a paradigm for understanding these biological processes. The RecA protein has been overproduced in E. coli and purified using a variety of purification schemes requiring multiple, time-intensive steps. The purification schemes share a dependence on appropriate RecA structure and/or function at one or more steps. In this report, we used a modified protein splicing element (intein) and a chitin-binding domain, fused to the C-terminus of RecA, to facilitate a one-step affinity purification of RecA protein without modification of the native protein sequence. Following the single chromatographic step, RecA protein that is greater than 95% physical purity at a concentration of greater than microM was obtained. The protein displays in vitro activities that are identical to those of protein isolated using classical procedures. The purification strategy described here promises to yield mutant RecA proteins in sufficient quantity for rigorous biophysical characterization without dependence on intrinsic RecA function.     

Mutagenic DNA repair in Escherichia coli. XIX. On the roles of RecA protein in ultraviolet light mutagenesis

An experimental system was used in which His+ mutations induced by ultraviolet light (UV) arise from non-photo-reversible photoproducts whereas lethality is largely determined by photoreversible photoproducts. By exposing a strain with a deletion through recA to light immediately after UV, it was possible to examine mutagenesis under conditions where survival was not significantly different from 100%. No UV mutagenesis was seen in the absence of RecA protein even though the rest of the SOS system was fully expressed due to the presence of a defective LexA repressor and the active carboxy-terminal fragment of UmuD was present as a result of an engineered plasmid-borne gene. We conclude that RecA protein has a third essential function if UV mutagenesis is to be detected in excision-deficient-bacteria. Another experiment showed that in exerting this function RecA protein does not need activation by pyrimidine dimers elsewhere on the genome, in contrast to its protein-cleavage mediation functions with LexA and UmuD proteins. RecA1730 protein blocked UV mutagenesis unless delayed photoreversal was given showing that the third function of RecA protein is not in the misincorporation step. It is therefore most likely to be in the bypass step where UmuD' and UmuC are postulated to act, although the possibility cannot be excluded that RecA protein is required for some other survival function distinct from translesion synthesis.     

Nature of the SOS mutator activity: genetic characterization of untargeted mutagenesis in Escherichia coli

Summary In Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA. Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA. The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected. This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e. non-targeted, replication errors. This recA730 mutator effect is suppressed by a mutation in the umuC gene. We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair. We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands. The recA730 mutation increases spontaneous mutagenesis of phage poorly. UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA ⁺ strains. This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation. Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations.     

Biochemical and biological function of Escherichia coli RecA protein: behavior of mutant RecA proteins

The recA protein of E coli participates in several diverse biological processes and promotes a variety of complex in vitro reactions. A careful comparison of the phenotypic behavior of E coli recA mutations to the biochemical properties of the corresponding mutant proteins reveals a close parallel both between recombination phenotype and DNA strand exchange and renaturation activities, and between inducible phenomena and repressor cleavage activity. The biochemical alterations manifest by the mutant recA proteins are reflected in the strength of their interaction with ssDNA. The defective mutant recA proteins fail to properly assume the high-affinity DNA-binding state that is characteristic of the wild-type protein and, consequently, form less stable complexes with DNA. The mutant proteins displaying an 'enhanced' activity bind ssDNA with approximately the same affinity as the wild-type protein but, due to altered protein-protein interactions, they associate more rapidly with ssDNA. These changes proportionately affect the ability of recA protein to compete with SSB protein, to interact with dsDNA, and, perhaps, to bind repressor proteins. In turn, the DNA strand exchange, DNA renaturation, and repressor cleavage activities mirror these modifications.     

Mechanism of SOS-induced targeted and untargeted mutagenesis in E. coli

This paper retraces the evolution of hypotheses concerning mechanisms of SOS induced mutagenesis. Moreover, it reports some recent data which support a new model for the mechanism of targeted and untargeted mutagenesis in E. coli. In summary, the SOS mutator effect, which is responsible for untargeted mutagenesis and perhaps for the misincorporation step in targeted mutagenesis, is believed to involve a fidelity function associated with DNA polymerase III and does not require the umuC gene product. umuC and umuD gene products are probably required specifically for elongation of DNA synthesis past blocking lesions, i.e. to allow mutagenic replication of damaged DNA.     

Role of Escherichia coli RecA protein in SOS induction and post-replication repair

The RecA protein of Escherichia coli plays a central role in DNA repair mechanisms. When it is incubated with single-stranded DNA and a nucleoside triphosphate, the purified RecA protein acts both by promoting cleavage of the LexA protein, the repressor of the SOS genes, and by catalyzing strand exchange between a variety of DNA molecules. A model for the regulation of the activity of the RecA protein in a cell exposed to a DNA damaging treatment is proposed.     

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.     

Inhibition of RecA protein function by the RdgC protein from Escherichia coli

The Escherichia coli RdgC protein is a potential negative regulator of RecA function. RdgC inhibits RecA protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA. The capacity of RecA to compete with RdgC is improved by the DinI protein. RdgC protein can inhibit DNA strand exchange catalyzed by RecA nucleoprotein filaments formed on single-stranded DNA by binding to the homologous duplex DNA and thereby blocking access to that DNA by the RecA nucleoprotein filaments. RdgC protein binds to single-stranded and double-stranded DNA, and the protein can be visualized on DNA using electron microscopy. RdgC protein exists in solution as a mixture of oligomeric states in equilibrium, most likely as monomers, dimers, and tetramers. This concentration-dependent change of state appears to affect its mode of binding to DNA and its capacity to inhibit RecA. The various species differ in their capacity to inhibit RecA function.     

Controlling and quantifying protein concentration in Escherichia coli

The cellular environment is dynamic and complex, involving thousands of different macromolecules with total concentrations of hundreds of grams per liter. However, most biochemistry is conducted in dilute buffer where the concentration of macromolecules is less than 10 g/L. High concentrations of macromolecules affect protein stability, function, and protein complex formation, but to understand these phenomena fully we need to know the concentration of the test protein in cells. Here, we quantify the concentration of an overexpressed recombinant protein, a variant of the B1 domain of protein G, in Tuner (DE3)? Escherichia coli cells as a function of inducer concentration. We find that the protein expression level is controllable, and expression saturates at over 2 mM upon induction with 0.4 mM isopropyl β‐d ‐thiogalactoside. We discuss the results in terms of what can and cannot be learned from in‐cell protein NMR studies in E. coli.     

Inhibition of pyrimidine dimer excision in ultraviolet-irradiated Escherichia coli overproducing RecA protein

Escherichia coli Br Hcr+ cells transformed with the recombinant multicopy plasmid pBR322 carrying recA gene contain increased amounts of RecA protein. When these cells were UV-irradiated, excision of pyrimidine dimers was reduced by about 50%. It is suggested that the damaged DNA strands may be coated with RecA protein which makes them insensitive to the action of the uvrABC excision nuclease.     

Analysis of the DNA binding site of Escherichia coli RecA protein

To investigate the DNA binding site of RecA protein, we constructed 15 recA mutants having alterations in the regions homologous to the other ssDNA binding proteins. The in vivo analyses showed that the mutational change at Arg²⁴³, Lys²⁴⁸, Tyr²⁶⁴, or simultaneously at Lys⁶ and Lys¹⁹, or Lys⁶ and Lys²³ caused severe defects in the recA functions, while other mutational changes did not. Purified RecA-K6A-K23A (Lys⁶ and Lys²³ changed to Ala and Ala, respectively) protein was indistinguishable from the wild-type RecA protein in its binding to DNA. However, the RecA-R243A (Arg²⁴³ changed to Ala) and RecA-Y264A (Tyr²⁶⁴ changed to Ala) proteins were defective in binding to both ss- and ds-DNA. In self-oligomerization property, RecA-R243A was proficient but RecA-Y264A was deficient, suggesting that the RecA-R243A protein had a defect in DNA binding site and the RecA-Y264A protein was defective in its interaction with the adjacent RecA molecule. The region of residues 243–257 including the Arg²⁴³ is highly homologous to the DNA binding motif in the ssDNA binding proteins, while the eukaryotic RecA homologues have a similar structure at the amino-terminal side proximal to the nucleotide binding core. The region of residues 243–257 would be a part of the DNA binding site. The other parts of this site would be the Tyr¹⁰³ and the region of residues 178–183, which were cross-linked to ssDNA. These three regions lie in a line in the crystal structure.