Role Of RecA Research Articles

DNA recombination and repair in Wolbachia: RecA and related proteins.

Wolbachia is an obligate intracellular bacterium that has undergone extensive genomic streamlining in its arthropod and nematode hosts. Because the gene encoding the bacterial DNA recombination/repair protein RecA is not essential in Escherichia coli, abundant expression of this protein in a mosquito cell line persistently infected with Wolbachia strain wStri was unexpected. However, RecA's role in the lytic cycle of bacteriophage lambda provides an explanation for retention of recA in strains known to encode lambda-like WO prophages. To examine DNA recombination/repair capacities in Wolbachia, a systematic examination of RecA and related proteins in complete or nearly complete Wolbachia genomes from supergroups A, B, C, D, E, F, J and S was undertaken. Genes encoding proteins including RecA, RecF, RecO, RecR, RecG and Holliday junction resolvases RuvA, RuvB and RuvC are uniformly absent from Wolbachia in supergroup C and have reduced representation in supergroups D and J, suggesting that recombination and repair activities are compromised in nematode-associated Wolbachia, relative to strains that infect arthropods. An exception is filarial Wolbachia strain wMhie, assigned to supergroup F, which occurs in a nematode host from a poikilothermic lizard. Genes encoding LexA and error-prone polymerases are absent from all Wolbachia genomes, suggesting that the SOS functions induced by RecA-mediated activation of LexA do not occur, despite retention of genes encoding a few proteins that respond to LexA induction in E. coli. Three independent E. coli accessions converge on a single Wolbachia UvrD helicase, which interacts with mismatch repair proteins MutS and MutL, encoded in nearly all Wolbachia genomes. With the exception of MutL, which has been mapped to a eukaryotic association module in Phage WO, proteins involved in recombination/repair are uniformly represented by single protein annotations. Putative phage-encoded MutL proteins are restricted to Wolbachia supergroups A and B and show higher amino acid identity than chromosomally encoded MutL orthologs. This analysis underscores differences between nematode and arthropod-associated Wolbachia and describes aspects of DNA metabolism that potentially impact development of procedures for transformation and genetic manipulation of Wolbachia.

An Epistasis Analysis of recA and recN in Escherichia coli K-12.

RecA is essential for double-strand-break repair (DSBR) and the SOS response in Escherichia coli K-12. RecN is an SOS protein and a member of the Structural Maintenance of Chromosomes family of proteins thought to play a role in sister chromatid cohesion/interactions during DSBR. Previous studies have shown that a plasmid-encoded recA4190 (Q300R) mutant had a phenotype similar to ∆recN (mitomycin C sensitive and UV resistant). It was hypothesized that RecN and RecA physically interact, and that recA4190 specifically eliminated this interaction. To test this model, an epistasis analysis between recA4190 and ∆recN was performed in wild-type and recBC sbcBC cells. To do this, recA4190 was first transferred to the chromosome. As single mutants, recA4190 and ∆recN were Rec+ as measured by transductional recombination, but were 3-fold and 10-fold decreased in their ability to do I-SceI-induced DSBR, respectively. In both cases, the double mutant had an additive phenotype relative to either single mutant. In the recBC sbcBC background, recA4190 and ∆recN cells were very UVS (sensitive), Rec-, had high basal levels of SOS expression and an altered distribution of RecA-GFP structures. In all cases, the double mutant had additive phenotypes. These data suggest that recA4190 (Q300R) and ∆recN remove functions in genetically distinct pathways important for DNA repair, and that RecA Q300 was not important for an interaction between RecN and RecA in vivorecA4190 (Q300R) revealed modest phenotypes in a wild-type background and dramatic phenotypes in a recBC sbcBC strain, reflecting greater stringency of RecA's role in that background.

Molecular and functional characterization of the Listeria monocytogenes RecA protein: Insights into the homologous recombination process.

The recombinases present in the all kingdoms in nature play a crucial role in DNA metabolism processes such as replication, repair, recombination and transcription. However, till date, the role of RecA in the deadly foodborne pathogen Listeria monocytogenes remains unknown. In this study, the authors show that L. monocytogenes expresses recA more than two-fold in vivo upon exposure to the DNA damaging agents, methyl methanesulfonate and ultraviolet radiation. The purified L. monocytogenes RecA protein show robust binding to single stranded DNA. The RecA is capable of forming displacement loop and hydrolyzes ATP, whereas the mutant LmRecAK70A fails to hydrolyze ATP, showing conserved walker A and B motifs. Interestingly, L. monocytogenes RecA and LmRecAK70A perform the DNA strand transfer activity, which is the hallmark feature of RecA protein with an oligonucleotide-based substrate. Notably, L. monocytogenes RecA readily cleaves L. monocytogenes LexA, the SOS regulon and protects the presynaptic filament from the exonuclease I activity. Altogether, this study provides the first detailed characterization of L. monocytogenes RecA and presents important insights into the process of homologous recombination in the gram-positive foodborne bacteria L. monocytogenes.

Investigating Intracellular RecA Concentrations in the Emerging Pathogen Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen responsible for many hospital‐acquired infections. A. baumannii is known for its ability to rapidly gain antibiotic resistances and survive desiccation. This ability to survive adverse conditions has been linked to A. baumannii's unique DNA‐damage response (DDR), which is modulated by the evolutionarily conserved recombinase, RecA. We have previously shown that in A. baumannii, RecA is needed to upregulate the expression of error‐prone DNA polymerase genes, which in turn results in higher frequency of mutagenesis.A. baumannii lacks a functional homologue for the highly conserved global Escherichia coli DDR transcriptional repressor, indicating that their DDRs are regulated differently. In contrast to E. coli, the DDR in A. baumannii is bimodal. That is, when DNA damage is induced and expression of DDR genes is tracked, two distinct subpopulations are evident, one with low expression of DDR genes and one with high. The two populations survive differently, suggesting that the bacterium can fall back on the population that does survive. The details of the mechanism underlying this regulation remain largely unknown. Our lab has recently shown that the recA gene is regulated by a 5′untranslated region that is important for both its stability and expression. Moreover, preliminary evidence suggests that differential recA expression is needed to maintain the bimodal nature of the DDR. Because of RecA's role in the DDR, we sought to compare the levels of RecA in A. baumannii and E. coli. We hypothesized that RecA intracellular concentration in A. baumannii would be lower than E. coli's, consistent with the differences in regulatory roles. To test this hypothesis, we performed a semiquantitative Western blot using anti‐E. coli RecA polyclonal antibody. We first purified His‐tagged RecA from each species. The purified proteins were then used to prepare a standard curve for the Western blot. As expected, we observed a difference in affinity of the anti‐recA antibody based on differences in the primary protein sequence of RecA from the two species. (A. baumannii and E. coli RecA are approximately 74% identical). Remarkably, and in agreement with our hypothesis, we find that the levels of basal intracellular RecA were at least three times lower in A. baumannii than in E. coli. This difference in RecA protein concentration may have profound effects on A. baumannii physiology and is consistent with the different regulatory role for RecA in A. baumannii. These results show that the intracellular RecA concentration in bacteria may provide insights into the regulation of the DDR and the mechanism by which A. baumannii is able to survive adverse conditions and readily acquire antibiotic resistances.Support or Funding InformationFunding was provided by the National Institute of General Medical Sciences (RO1GM088230 to Veronica Godoy).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RecA: a universal drug target in pathogenic bacteria.

The spread of bacterial infectious diseases due to the development of resistance to antibiotic drugs in pathogenic bacteria is an emerging global concern. Therefore, the efficacious management and prevention of bacterial infections are major public health challenges. RecA is a pleiotropic recombinase protein that has been demonstrated to be implicated strongly in the bacterial drug resistance, survival and pathogenicity. In this minireview, RecA's role in the development of antibiotic resistance and its potential as an antimicrobial drug target are discussed.

Role of RecA and the SOS Response in Thymineless Death in Escherichia coli

Thymineless death (TLD) is a classic and enigmatic phenomenon, documented in bacterial, yeast, and human cells, whereby cells lose viability rapidly when deprived of thymine. Despite its being the essential mode of action of important chemotherapeutic agents, and despite having been studied extensively for decades, the basic mechanisms of TLD have remained elusive. In Escherichia coli, several proteins involved in homologous recombination (HR) are required for TLD, however, surprisingly, RecA, the central HR protein and activator of the SOS DNA–damage response was reported not to be. We demonstrate that RecA and the SOS response are required for a substantial fraction of TLD. We show that some of the Rec proteins implicated previously promote TLD via facilitating activation of the SOS response and that, of the roughly 40 proteins upregulated by SOS, SulA, an SOS–inducible inhibitor of cell division, accounts for most or all of how SOS causes TLD. The data imply that much of TLD results from an irreversible cell-cycle checkpoint due to blocked cell division. FISH analyses of the DNA in cells undergoing TLD reveal blocked replication and apparent DNA loss with the region near the replication origin underrepresented initially and the region near the terminus lost later. Models implicating formation of single-strand DNA at blocked replication forks, a SulA-blocked cell cycle, and RecQ/RecJ-catalyzed DNA degradation and HR are discussed. The data predict the importance of DNA damage-response and HR networks to TLD and chemotherapy resistance in humans.

A novel role for RecA under non-stress: promotion of swarming motility in Escherichia coli K-12

BackgroundBacterial motility is a crucial factor in the colonization of natural environments. Escherichia coli has two flagella-driven motility types: swimming and swarming. Swimming motility consists of individual cell movement in liquid medium or soft semisolid agar, whereas swarming is a coordinated cellular behaviour leading to a collective movement on semisolid surfaces. It is known that swimming motility can be influenced by several types of environmental stress. In nature, environmentally induced DNA damage (e.g. UV irradiation) is one of the most common types of stress. One of the key proteins involved in the response to DNA damage is RecA, a multifunctional protein required for maintaining genome integrity and the generation of genetic variation.ResultsThe ability of E. coli cells to develop swarming migration on semisolid surfaces was suppressed in the absence of RecA. However, swimming motility was not affected. The swarming defect of a ΔrecA strain was fully complemented by a plasmid-borne recA gene. Although the ΔrecA cells grown on semisolidsurfaces exhibited flagellar production, they also presented impaired individual movement as well as a fully inactive collective swarming migration. Both the comparative analysis of gene expression profiles in wild-type and ΔrecA cells grown on a semisolid surface and the motility of lexA1 [Ind-] mutant cells demonstrated that the RecA effect on swarming does not require induction of the SOS response. By using a RecA-GFP fusion protein we were able to segregate the effect of RecA on swarming from its other functions. This protein fusion failed to regulate the induction of the SOS response, the recombinational DNA repair of UV-treated cells and the genetic recombination, however, it was efficient in rescuing the swarming motility defect of the ΔrecA mutant. The RecA-GFP protein retains a residual ssDNA-dependent ATPase activity but does not perform DNA strand exchange.ConclusionThe experimental evidence presented in this work supports a novel role for RecA: the promotion of swarming motility. The defective swarming migration of ΔrecA cells does not appear to be associated with defective flagellar production; rather, it seems to be associated with an abnormal flagellar propulsion function. Our results strongly suggest that the RecA effect on swarming motility does not require an extensive canonical RecA nucleofilament formation. RecA is the first reported cellular factor specifically affecting swarming but not swimming motility in E. coli. The integration of two apparently disconnected biologically important processes, such as the maintenance of genome integrity and motility in a unique protein, may have important evolutive consequences.

Critical role of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of rejoining following γ-radiation-induced damage to pMTa4 DNA in E. coli

Purpose: This study was undertaken to understand the roles of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of the process following exposure of E. coli to γ-radiation in vivo.Materials and methods: A plasmid DNA construct, pMTa4, was transformed into isogenic repair proficient (wild) and deficient (recF and recA) E. coli strains and γ-irradiated up to 30 Gy in vivo. The plasmid DNA was isolated under repair non-permissive (R−)and permissive (R+) conditions and analyzed by gel electrophoresis for the yields of single strand breaks (SSB) and double strand breaks (DSB) and their repair. The clonogenic survival of the E. coli was also recorded. The effects of γ-irradiation on recA reconstituted with cell free extract of wild strain or ultra-violet (UV)-irradiation were also monitored.Results: None of the strains used in this investigation showed effects of radiation-induced oxidative base damage. The dose dependent increase in SSB and DSB on pMTa4 in wild and recF mutants in R− condition were abolished upon repair incubation. The recA mutant exhibited a disturbed yield of SSB and DSB along with formation of γ-radiation-induced ‘ladder’. The ‘ladder’ was not observed after repair incubation, UV-irradiation or γ-irradiation in presence of cell-free extract of wild strain. The survival of recA mutants was seriously compromised.Conclusions: Wild, recF and recA strains of E. coli could repair γ-irradiation-induced oxidative damage to base or nucleotide (NT) in vivo. In absence of either RecA or RecF proteins, efficiency of rejoining of strand went down; RecA proteins seemed more critical than RecF in this. High fidelity or correct rejoining of strand breaks, on the other hand, seemed to require simultaneous presence of both RecA and RecF proteins.

Genotoxicity of water extracts from the River Yamuna at Mathura, India.

Water samples were collected from the River Yamuna at Mathura, India, and concentrated by using XAD resins (Amberlite XAD-4 and XAD-8) and liquid-liquid extraction procedures. The genotoxicities of the extracted water samples were evaluated by the Ames Salmonella/mammalian microsome test, DNA repair of defective mutants, and bacteriophage lambda systems. The results of the Salmonella test demonstrated that the XAD-concentrated water samples had maximum mutagenicity with the TA98 strain, both with and without metabolic activation. The XAD-concentrated water samples collected in the summer showed maximum mutagenic responses compared with those collected in other seasons, whereas the liquid-liquid extracted samples exhibited maximum mutagenic activity during the postmonsoon season. The damage brought about during DNA repair of defective mutants in the presence of XAD-concentrated water samples was found to be remarkably high compared with the liquid-liquid extracted water samples at a dose level of 20 microL/mL of culture. All the mutants invariably exhibited significant decline in their colony-forming units compared with their isogenic wild-type counterparts. Survival was decreased by 86.7% and 65.1% in the polA(-) strain after 6 h of treatment with XAD-concentrated and liquid-liquid extracted water samples, respectively. A significant decrease in the survival of bacteriophage lambda was also observed when treated with test samples. The damage was more pronounced in lexA mutants when the phage was treated with XAD-concentrated samples. The recA, lexA, and polA mutants of E. coli K-12 were found to be sensitive to the test samples, suggesting damage to the DNA of exposed cells as well as to the role of recA(+), lexA(+), and polA(+) genes in coping with the hazardous effect of the pollutants. The results demonstrated substantial genotoxicity and mutagenicity in the water samples tested.

Alteration of cell morphology and viability in a recA mutant of Streptococcusthermophilus upon induction of heat shock and nutrient starvation

We identified the recA gene of the moderately thermophilic bacterium Streptococcus thermophilus and investigated the role of its product in the adaptation to heat shock and nutrient starvation. Expression of recA was required for optimal viability and normal cell morphology upon induction of both stresses. Normal induction of GroEL and ClpL in a recA knock-out mutant suggests that the RecA role in heat shock and nutrient starvation response of S. thermophilus is independent from the intracellular accumulation of these stress-specific chaperones.

A Molecular Model for RecA-Promoted Strand Exchange via Parallel Triple-Stranded Helices

A number of studies have concluded that strand exchange between a RecA-complexed DNA single strand and a homologous DNA duplex occurs via a single-strand invasion of the minor groove of the duplex. Using molecular modeling, we have previously demonstrated the possibility of forming a parallel triple helix in which the single strand interacts with the intact duplex in the minor groove, via novel base interactions (Bertucat et al., J. Biomol. Struct. Dynam. 16:535–546). This triplex is stabilized by the stretching and unwinding imposed by RecA. In the present study, we show that the bases within this triplex are appropriately placed to undergo strand exchange. Strand exchange is found to be exothermic and to result in a triple helix in which the new single strand occupies the major groove. This structure, which can be equated to so-called R-form DNA, can be further stabilized by compression and rewinding. We are consequently able to propose a detailed, atomic-scale model of RecA-promoted strand exchange. This model, which is supported by a variety of experimental data, suggests that the role of RecA is principally to prepare the single strand for its future interactions, to guide a minor groove attack on duplex DNA, and to stabilize the resulting, stretched triplex, which intrinsically favors strand exchange. We also discuss how this mechanism can incorporate homologous recognition.

Involvement of recF, recO, and recR genes in UV-radiation mutagenesis of Escherichia coli.

The recF, recO, and recR genes were originally identified as those affecting the RecF pathway of recombination in Escherichia coli cells. Several lines of evidence suggest that the recF, recO, and recR genes function at the same step of recombination and postreplication repair. In this work, we report that null mutations in recF, recO, or recR greatly reduce UV-radiation mutagenesis (UVM) in an assay for reversion from a Trp- (trpE65) to a Trp+ phenotypes. Introduction of the defective lexA51 mutation [lexA51(Def)] and/or UmuD' into recF, recO, and recR mutants failed to restore normal UVM in the mutants. On the other hand, the presence of recA2020, a suppressor mutation for recF, recO, and recR mutations, restored normal UVM in recF, recO, and recR mutants. These results indicate an involvement of the recF, recO, and recR genes and their products in UVM, possibly by affecting the third role of RecA in UVM.

The recA gene of Lactococcus lactis: characterization and involvement in oxidative and thermal stress.

The role of recA in Lactococcus lactis, a microaerophilic fermenting organism, was examined by constructing a recA-disrupted strain. This single alteration had a surprisingly pleiotropic effect. In addition to its roles in homologous recombination and DNA repair, recA is also involved in responses to oxygen and heat stresses. We found that oxygen stress induced by aeration causes reductions in growth and stationary-phase survival of the recA strain. Toxicity is a consequence of hydroxyl radical production via the Fenton Reaction and is alleviated by catalase or Ferrozine addition. These results suggest that oxygen radicals are not efficiently eliminated and accumulate in lactococcal cultures, and that RecA is needed to deal with the damage they incur. Unexpectedly, thermal stress arrested growth of the recA strain. Immunological data indicate that the recA mutant is deficient in heat-shock proteins DnaK, GroEL, and GrpE. Poor growth at elevated temperature is therefore due to a diminished heat-shock response in the recA strain. In contrast, levels of a novel heat-shock protein, HfIB, are elevated. In Escherichia coli, HfIB downregulates the heat-shock response by promoting degradation of the transcription factor sigma 32. We propose that recA regulates the heat-shock response via HfIB. This work provides the first evidence showing that two major pathways of stress response, induced by heat shock and DNA damage, are interactive.

Structure and function of RecA-DNA complexes

While the E. coli RecA protein has been the most intensively studied enzyme of homologous recombination, the unusual RecA-DNA filament has stood alone until very recently. It now appears that this protein is part of a universal family that spans all of biology, and the filament that is formed by the protein on DNA is a universal structure. With RecA's role in recombination given new and greatly increased significance, we focus in this review on the energetics of the RecA-mediated strand exchange and the relation between the energetics and recombination spanning heterologous inserts.

Requirement for the polymerization and 5'-->3' exonuclease activities of DNA polymerase I in initiation of DNA replication at oriK sites in the absence of RecA in Escherichia coli rnhA mutants.

In previous studies, we found that the requirement for RecA protein in constitutive stable DNA replication (cSDR) can be bypassed by derepression of the LexA regulon and that DNA polymerase I (DNA PolI) is essential for this Rip (RecA-independent process) pathway of cSDR (Y. Cao, R. R. Rowland, and T. Kogoma, J. Bacteriol. 175:7247-7253, 1993). In this study, the role of DNA PolI in the Rip pathway was further examined. By using F' plasmids carrying different parts of the polA gene, a series of complementation tests was carried out to investigate the requirement for the three enzymatic activities, polymerization, 3'-->5' exonuclease, and 5'-->3' exonuclease activities, of DNA PolI. The result indicated that both the 5'-->3' exonuclease and polymerization activities of DNA PolI are essential for bypassing the requirement for RecA in cSDR but that the 3'-->5' exonuclease activity can be dispensed with. Complementation experiments with rat DNA Pol beta also supported the hypothesis that a nick translation activity is probably involved in cSDR in the absence of RecA. An analysis of DNA synthesis suggested that DNA PolI is involved in the initiation but not the elongation stage of cSDR. Moreover, the dnaE293(Ts) mutation was shown to render the bypass replication temperature sensitive despite the presence of active DNA PolI, suggesting that DNA PolIII is responsible for the elongation stage of the Rip pathway. A model which describes the possible roles of RecA in cSDR and the possible function of DNA PolI in the Rip pathway is proposed.

Cross-adaptive response in Escherichia coli caused by pretreatment with H2O2 against formaldehyde and other aldehyde compounds

A cross-adaptive response (CAR), defined as a reduction of the effects of an agent by pretreatment with another agent, was demonstrated when E. coli WP2 cells were pretreated with hydrogen peroxide (H2O2) followed by challenging treatment with aldehyde compounds. Pretreatment with a sublethal dose (60 μM) of H2O2 for 30 min made WP2 cells resistant to the killing effects of formaldehyde (FA), and 4 other mutagenic aldehydes: glutaraldehyde, glyoxal, methyl glyoxal and chloroacetaldehyde. CAR was also observed in WP2uvrA (uvrA−) and ZA12 (umuC−) cells, but not in ZA60 (recA−) and CM561 (lexA− (ind−)) cells. A role of recA and lexA in CAR was further suggested by the lack of β-galactosidase induction in recA− and lexA− cells by H2O2. CAR and β-galactosidase induction, however, were found to be separate events since CAR was recovered by introducing the recA+ gene into lexA− cells, but no induction of β-galactosidase by H2O2 was observed in cells with the same gene transfer. These results suggest that H2O2 has the capacity to induce a function which reduces the killing effects of aldehydes, and the function is controlled by the recA gene without involvement of SOS response.

Requirements for bypass of UV-induced lesions in single-stranded DNA of bacteriophage phi X174 in Salmonella typhimurium.

According to the current model for mutagenic bypass of UV-induced lesions, efficient bypass requires three proteins: activated RecA (RecA*) and either activated UmuD (UmuD') and UmuC or their plasmid-encoded analogues, MucA' and MucB. RecA* aids synthesis of UmuD' and UmuC (and MucA'/MucB) at two levels: by inactivation of the LexA transcriptional repressor of these genes and by cleavage of UmuD (and MucA) to produce the active fragments, UmuD' (MucA'). A third role for RecA is revealed when these two roles are otherwise satisfied in a suitably engineered strain. An often-suggested possible role for RecA in bypass is inhibition of editing by the epsilon subunit of DNA polymerase III. Here, by demonstrating that elimination of epsilon by deletion of its gene, dnaQ, does not relieve the requirement for the third function of RecA, we show that RecA must perform some function other than, or in addition to, inhibition of epsilon. We also show that elimination of epsilon does not relieve the requirement for either Muc protein. Moreover, we observed reactivation of irradiated phi X174 in unirradiated cells expressing MucA' and MucB. This finding makes it unlikely that the additional role of recA involves derepression of an unidentified gene or cleavage of an unidentified protein and makes it more likely that RecA participates directly in bypass.

Mutagenic activity of certain synthetic steroids: structural requirement for the mutagenic activity in Salmonella and E. coli

Eight steroids, structurally related to cholesterol, were tested for mutagenic activity in the Ames tester strains. All the test compounds were mutagenic without metabolic activation, although metabolic activation markedly enhanced their activity. A significant decrease in the survival of the radiation-sensitive mutants recA and lexA of Escherichia coli was observed as compared to their wild-type counterpart in the presence of the steroids. The role of recA and lexA genes gains further support from the λ prophage induction in the lysogen as well as with Salmonella strains triggering the error-prone SOS response. Structural features which appear to be essential for mutagenic activity in these strains of the steroids are (1) reactive thio, sulfonyl or sulfinyl groups at the 6 position and (2) a halogen group at the 3 position of the steroidal nucleus. The mutagenicity appears to involve the formation of H 2O 2 as well as superoxide and hydroxyl radicals.

Genetic control of AAF-induced mutagenesis at alternating GC sequences: An additional role for RecA

In a previous study, the forward mutation spectrum induced by the chemical carcinogen N-acetoxy-N-2-acetylaminofluorene was determined (Koffel-Schwartz et al. 1984). It was found that 90% of the induced mutations are frameshift mutations located within specific sequences (mutation hot spots). Two classes of mutation hot spots were found: (i) -1 frameshift mutations occurring within runs of guanines (i.e. GGGG----GGG; (ii) -2 frameshift mutations occurring within the NarI recognition sequence (GGCGCC----GGCC). In the present work, we further investigate the genetic requirements of these frameshift events by using specific reversion assays. Like UV-induced mutagenesis, frameshift mutations occurring within runs of G's (also referred to as the "slippage pathway") require the activated form of the RecA protein (RecA*). On the other hand, frameshift mutations occurring at the NarI site (the "NarI mutation pathway") require a LexA-controlled function(s) that is not UmuDC. The LexA-controlled gene(s) that is (are) involved in this pathway remain to be identified. Moreover, this pathway does not require RecA* for the proteolytic processing of a protein other than LexA (like the cleavage of UmuD in UV-induced mutagenesis). An "additional" role of RecA can be defined as follows: (i) The non-activated form of the RecA protein acts as an inhibitor in the NarI mutation pathway. (ii) This inhibition is relieved upon activation of RecA by UV irradiation of the bacteria. (iii) A recA deletion mutant is totally proficient in the NarI mutation pathway provided the SOS system is derepressed [lexA (Def) allele]. Therefore, RecA does not actively participate in the fixation of the mutation. A molecular model for this "additional" role of RecA is proposed.

UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA.

The mutation rate of Escherichia coli increases approximately 100-fold after treatment with replication-inhibiting agents such as UV light. This enhanced mutation rate requires the action of the UmuD and UmuC proteins, which are induced as part of the SOS response to DNA damage. To initiate a biochemical characterization of the role of these proteins, we have developed a plasmid system that gives efficient expression of the umuD and umuC genes. The umuD and umuC genes were placed under the control of a regulated phage lambda PL promoter and a synthetic ribosome-binding site, and the distance to the UmuD start was adjusted to maximize gene expression. Starting from this overproduction system, we have purified the UmuD protein and studied its interaction with RecA. The SOS response is turned on by the capacity of RecA protein to mediate cleavage of the LexA repressor for SOS-controlled operons. Others have shown that UmuD exhibits sequence homology to LexA around the cleavage site, suggesting a possible cleavage reaction for UmuD. We show that RecA mediates cleavage of UmuD, probably at this site. As with LexA, UmuD also undergoes a self-cleavage reaction. We infer that RecA-mediated cleavage of UmuD is another role for RecA in SOS mutagenesis, probably activating UmuD for its mutagenic function.