Induction of the SOS Response in Ultraviolet-Irradiated Escherichia coli Analyzed by Dynamics of LexA, RecA and SulA Proteins

The SOS response in Escherichia coli is induced after DNA-damaging treatments including ultraviolet light. Regulation of the SOS response is accomplished through specific interaction of the two SOS regulator proteins, LexA and RecA. In ultraviolet light-treated cells, nucleotide excision repair is the major system that removes the induced lesions from the DNA. Here, induction of the SOS response in Escherichia coli with normal and impaired excision repair function is studied by simulation of intracellular levels of regulatory LexA and RecA proteins, and SulA protein. SulA protein is responsible for SOS-inducible cell division inhibition. Results of the simulations show that nucleotide excision repair influences time-courses of LexA, RecA and SulA induction by modulating the dynamics of RecA protein distribution between its normal and SOS-activated forms.     

UvrD limits the number and intensities of RecA-green fluorescent protein structures in Escherichia coli K-12

RecA is important for recombination, DNA repair, and SOS induction. In Escherichia coli, RecBCD, RecFOR, and RecJQ prepare DNA substrates onto which RecA binds. UvrD is a 3'-to-5' helicase that participates in methyl-directed mismatch repair and nucleotide excision repair. uvrD deletion mutants are sensitive to UV irradiation, hypermutable, and hyper-rec. In vitro, UvrD can dissociate RecA from single-stranded DNA. Other experiments suggest that UvrD removes RecA from DNA where it promotes unproductive reactions. To test if UvrD limits the number and/or the size of RecA-DNA structures in vivo, an uvrD mutation was combined with recA-gfp. This recA allele allows the number of RecA structures and the amount of RecA at these structures to be assayed in living cells. uvrD mutants show a threefold increase in the number of RecA-GFP foci, and these foci are, on average, nearly twofold higher in relative intensity. The increased number of RecA-green fluorescent protein foci in the uvrD mutant is dependent on recF, recO, recR, recJ, and recQ. The increase in average relative intensity is dependent on recO and recQ. These data support an in vivo role for UvrD in removing RecA from the DNA.     

Overexpression of bacterial RecA protein stimulates homologous recombination in somatic mammalian cells

The pairing of homologous molecules and strand exchange is a key event in homologous recombination promoted by RecA protein in Escherichia coli. Structural homologs of RecA are widely distributed in eukaryotes including mouse and man. As has been shown, human HsRad51 protein is not only structural but also functional homolog of RecA. The question arises whether the bacterial functional homolog of Rad51 can function in mammalian cells and increase the frequency of the homologous recombination. To investigate possible effects of bacterial RecA protein on the frequency of homologous recombination in mammalian cells, the E. coli RecA protein fused with a nuclear location signal from the large T antigen of simian virus 40 was overexpressed in the mouse F9 teratocarcinoma cells. We found that the frequency of gene targeting at the hprt locus was 10-fold increased in the mouse cells expressing the nucleus-targeted RecA protein. Southern blot analysis of individual clones that were generated by targeting recombination revealed predicted type of alterations in hprt gene. The data indicate that the bacterial nucleus-targeted RecA protein can stimulate homologous recombination in mammalian cells.     

RecA Expression in Response to Solar UVR in the Marine Bacterium Vibrio natriegens

Solar ultraviolet radiation may produce daily stress on marine and estuarine communities as cells are damaged and repair that damage. Reduction in the earth's stratospheric ozone layer has increased awareness of the potential effects that ultraviolet radiation may have in the environment, including how marine bacteria respond to changes in solar radiation. We examined the use of the bacterial RecA protein as an indicator of the potential of bacteria to repair DNA damage caused by solar UV irradiation using the marine bacterium Vibrio natriegens as a model. RecA is universally present in bacteria and is a regulator protein for the so-called Dark Repair Systems, which include excision repair, postreplication recombinational repair, and mutagenic or SOS repair. Solar UVB and UVA both reduced V. natriegens viability in seawater microcosms. After exposure to unfiltered solar radiation or radiation in which UVB was blocked, survival dropped below 1%, whereas visible light from which UVA and UVB had been filtered had no effect on survival. Using a RecA-specific antibody for detection, RecA protein was induced by solar radiation in a diel pattern in marine microcosms conducted in the Gulf of Mexico. Peak induction was observed at dusk each day. Although RecA expression was correlated with the formation of UVB-induced cyclobutyl pyrimidine dimers, longer wavelength UVA radiation also induced recA gene expression. Our results demonstrate that RecA-regulated, light-independent repair is an important component in the ability of marine bacteria to survive exposure to solar ultraviolet radiation and that RecA expression is a useful monitor of bacterial repair after exposure to solar UVR.     

Excision and Transposition of Tn5 as an Sos Activity in Escherichia Coli

Excision and transposition of the Tn5 element in Escherichia coli ordinarily appear to occur by recA-independent mechanisms. However, recA(Prtc) genes, which encode RecA proteins that are constitutively activated to the protease state, greatly enhanced excision and transposition; both events appeared to occur concomitantly and without destruction of the donor DNA. The recombinase function of the RecA protein was not required. Transposition was accompanied by partial, and occasionally full, restoration of the functional integrity of the gene vacated by the excised Tn5. The stimulation of transposition was inhibited by an uncleavable LexA protein and was strongly enhanced by an additional role of the RecA(Prtc) protein besides its mediation of LexA cleavage. To account for the enhanced transposition, we suggest that (i) there may be a LexA binding site within the promoter for the IS50 transposase, (ii) activated RecA may cleave the IS50 transposition inhibitor, and (iii) the transposase may be formed by RecA cleavage of a precursor molecule.     

Molecular cloning and characterization of novel tissue-specific isoforms of the human vacuolar H(+)-ATPase C,G and d subunits,and their evaluation in autosomal recessive distal renal tubular acidosis

Protein splicing involves the self-catalyzed excision of an intervening sequence, the intein, from a precursor protein, with the concomitant ligation of the flanking extein sequences to yield a new polypeptide. The ability of inteins to promote protein splicing even when inserted into a foreign context has facilitated the study of the modulation of protein splicing. In this paper, we describe an in vivo screening system for the isolation of mutations or inhibitors that interfere with protein splicing mediated by the RecA intein of Mycobacterium tuberculosis. It involves the activation of the cytotoxic CcdB protein by protein splicing, such that host cells survive in the presence of inducer only when protein splicing is blocked. The coding sequence for the RecA intein was inserted in-frame into the polylinker region of an inducible lacZ alpha-ccdB fusion vector, leading to inactivation of the CcdB toxin unless the intein is excised by protein splicing. Depending on the objective of the screening procedure, its stringency can be modified by altering the level of expression of the intein-CcdB fusion protein. To induce large amounts of CcdB fusion proteins, the fusion protein is expressed from a high-copy-number plasmid. Such a screening system detects even low levels of protein splicing and we have used it to show that protein splicing of the RecA intein is compatible with any amino acid in the extein position adjacent to the N-terminal splice junction. In order to search for protein splicing inhibitors, which may attenuate protein splicing by less than an order of magnitude, we have also constructed a low-copy-number intein-CcdB plasmid so that the host cells can survive when splicing of the expressed CcdB fusion protein is only moderately suppressed. We anticipate that the CcdB-based in vivo screening system will find uses in the analysis of structural and mechanistic aspects of protein splicing.     

Targeting of a functional Escherichia coli RecA protein to the nucleus of plant cells

We have characterised a RecA protein fused to the simian virus 40 large T nuclear-localisation signal. The fusion protein was targeted to the nucleus in transgenic tobacco plants with high efficiency. By contrast, authentic RecA was not enriched in the nuclei of plant cells expressing comparable amounts of protein. For detailed characterisation of the strand-exchange activity of the nuclear-targeted RecA protein, a nearly identical protein was expressed in Escherichia coli and purified to homogeneity. This protein was found to bind to single-stranded DNA with the same stoichiometry and to promote the exchange of homologous DNA strands with the same kinetics as authentic RecA. It was concluded that the amino-terminal modification did not alter any of the essential properties of RecA and that the fusion protein is a fully functional strand-exchange protein. However, the ATPase activity of this protein was 20 times greater than that of RecA in the absence of single-stranded DNA. As with RecA, this activity was further stimulated by the addition of single-stranded DNA. Since ATPase activity is correlated with the ability of RecA to assume its high affinity state for DNA, the nuclear-targeted RecA protein might be regarded as a constitutively stimulated RecA variant, fully functional in promoting homologous recombination.     

Targeting of a functional Escherichia coli RecA protein to the nucleus of plant cells

 We have characterised a RecA protein fused to the simian virus 40 large T nuclear-localisation signal. The fusion protein was targeted to the nucleus in transgenic tobacco plants with high efficiency. By contrast, authentic RecA was not enriched in the nuclei of plant cells expressing comparable amounts of protein. For detailed characterisation of the strand-exchange activity of the nuclear-targeted RecA protein, a nearly identical protein was expressed in Escherichia coli and purified to homogeneity. This protein was found to bind to single-stranded DNA with the same stoichiometry and to promote the exchange of homologous DNA strands with the same kinetics as authentic RecA. It was concluded that the amino-terminal modification did not alter any of the essential properties of RecA and that the fusion protein is a fully functional strand-exchange protein. However, the ATPase activity of this protein was 20 times greater than that of RecA in the absence of single-stranded DNA. As with RecA, this activity was further stimulated by the addition of single-stranded DNA. Since ATPase activity is correlated with the ability of RecA to assume its high affinity state for DNA, the nuclear-targeted RecA protein might be regarded as a constitutively stimulated RecA variant, fully functional in promoting homologous recombination. Received: 29 July 1996 / Accepted: 24 September 1996     

Biochemical basis of the constitutive coprotease activity of RecA P67W protein

The mutation of Pro67 to Trp (P67W) in the Escherichia coli RecA protein results in reduced recombination and constitutive coprotease phenotypes. We examined the biochemical properties of this mutant in an effort to understand these altered behaviors. We find that RecA P67W protein can access single-stranded DNA (ssDNA) binding sites within regions of secondary structure more effectively than wild-type protein, and binding to duplex DNA is both faster and more extensive as well. This mutant is also more effective than wild-type RecA protein in displacing SSB protein from ssDNA. An enhancement in SSB protein displacement has been shown previously for RecA441, RecA730, and RecA803 proteins, and similarly, this improved ability to displace SSB protein for RecA P67W protein correlates with an increased rate of association with ssDNA. As for the aforementioned mutant RecA proteins, we expect that this enhanced activity will allow RecA P67W protein to bind ssDNA naturally occurring in undamaged cells and to constitutively induce the SOS response. The DNA strand exchange activity of RecA P67W protein is also altered. Although the rate of duplex DNA uptake into joint molecules is increased compared to that of wild-type RecA protein, the resolution to the nicked circular dsDNA product is reduced. We suggest that either a limited amount of DNA strand reinvasion or a defect in DNA heteroduplex extension is responsible for the impaired recombination ability of this mutant protein.     

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     

Distinguishing characteristics of hyperrecombinogenic RecA protein from Pseudomonas aeruginosa acting in Escherichia coli

In Escherichia coli, a relatively low frequency of recombination exchanges (FRE) is predetermined by the activity of RecA protein, as modulated by a complex regulatory program involving both autoregulation and other factors. The RecA protein of Pseudomonas aeruginosa (RecA(Pa)) exhibits a more robust recombinase activity than its E. coli counterpart (RecA(Ec)). Low-level expression of RecA(Pa) in E. coli cells results in hyperrecombination (an increase of FRE) even in the presence of RecA(Ec). This genetic effect is supported by the biochemical finding that the RecA(Pa) protein is more efficient in filament formation than RecA K72R, a mutant protein with RecA(Ec)-like DNA-binding ability. Expression of RecA(Pa) also partially suppresses the effects of recF, recO, and recR mutations. In concordance with the latter, RecA(Pa) filaments initiate recombination equally from both the 5' and 3' ends. Besides, these filaments exhibit more resistance to disassembly from the 5' ends that makes the ends potentially appropriate for initiation of strand exchange. These comparative genetic and biochemical characteristics reveal that multiple levels are used by bacteria for a programmed regulation of their recombination activities.     

Roles of RecA protease and recombinase activities of Escherichia coli in spontaneous and UV-induced mutagenesis and in Weigle repair.

The RecA protein has a second, direct role in the mutagenesis of Escherichia coli and bacteriophage lambda in addition to its first, indirect role of inducing the SOS system by enhancing the proteolytic cleavage of the LexA repressor protein. The need for RecA protease and recombinase functions in the direct role was examined in cells containing split-phenotype RecA mutations, in the absence of LexA protein. Spontaneous mutation of E. coli (his----his+) required both the protease and recombinase activities. The mutation frequency increased with increasing RecA protease strength. In contrast, UV-induced mutation of E. coli required only the RecA protease activity. Weigle repair and mutation of UV-irradiated phage S13 required only RecA protease activity, and even weak activity was highly effective; RecA recombinase activity was not required. RecA+ protein inhibited RecA (Prtc [protease constitutive] Rec+) protein in effecting spontaneous mutation of E. coli. We discuss the nature of the direct role of the RecA protein in spontaneous mutation and in repair and mutagenesis of UV-damaged DNA and also the implications of our results for the theory that SOS-mutable cryptic lesions might be responsible for the enhanced spontaneous mutation in Prtc Rec+ strains.     

In UV-irradiated Escherichia coli PQ35 overproducing the RecA protein, expression of the sfiA gene and dimer excision are alleviated

Summary Escherichia coli PQ 35 cells carrying thesfiA-:lacZ operon fusion were transformed either with a multicopy plasmid containing therecA gene (pHSG262recA) or with a multicopy plasmid alone (pHSG262). Both transformants were UV irradiated. Then induction of thesfiA gene and dimer excision were followed. Amplification of therecA gene partly inhibited bothsfiA gene induction and dimer excision. The following interpretation of this phenomenon is proposed. When the RecA protein is in bundance, pyrimidine dimers are quickly masked by it. The masked dimers are less efficiently distinguished by excision nuclease and do not provide the induction signal. Due to this, induction of thesfiA gene as well as dimer excision are inhibited early.     

Cloning of the recA gene from a free-living leptospire and distribution of RecA-like protein among spirochetes

A recombinant plasmid carrying the recA gene of Leptospira biflexa serovar patoc was isolated from a cosmid library of genomic DNA by complementation of an Escherichia coli recA mutation. The cloned serovar patoc recA gene efficiently restored resistance to UV radiation and methyl methanesulfonate. Recombination proficiency was also restored, as measured by the formation of Lac+ recombinants from duplicated mutant lacZ genes. Additionally, the cloned recA gene increased the spontaneous and mitomycin C-induced production of lambda phage in lysogens of an E. coli recA mutant. The product of the cloned recA gene was identified in maxicells as a polypeptide with an Mr of 43,000. Antibodies prepared against the E. coli RecA protein cross-reacted with the serovar patoc RecA protein, indicating structural conservation. Southern hybridization data showed that the serovar patoc recA gene has diverged from the recA gene of L. interrogans, Leptonema illini, and E. coli. With the exception of the RecA protein of L. interrogans serovar hardjo, the RecA protein of the Leptospira serovars and L. illini were synthesized at elevated levels following treatment of cells with nalidixic acid. The level of detectable RecA correlated with previous studies demonstrating that free-living cells of L. biflexa serovars and L. illini were considerably more resistant to DNA-damaging agents than were those of parasitic L. interrogans serovars. RecA protein was not detected in cells of virulent Treponema pallidum or Borrelia burgdorferi.     

Cloning of the recA gene from a free-living leptospire and distribution of RecA-like protein among spirochetes.

A recombinant plasmid carrying the recA gene of Leptospira biflexa serovar patoc was isolated from a cosmid library of genomic DNA by complementation of an Escherichia coli recA mutation. The cloned serovar patoc recA gene efficiently restored resistance to UV radiation and methyl methanesulfonate. Recombination proficiency was also restored, as measured by the formation of Lac+ recombinants from duplicated mutant lacZ genes. Additionally, the cloned recA gene increased the spontaneous and mitomycin C-induced production of lambda phage in lysogens of an E. coli recA mutant. The product of the cloned recA gene was identified in maxicells as a polypeptide with an Mr of 43,000. Antibodies prepared against the E. coli RecA protein cross-reacted with the serovar patoc RecA protein, indicating structural conservation. Southern hybridization data showed that the serovar patoc recA gene has diverged from the recA gene of L. interrogans, Leptonema illini, and E. coli. With the exception of the RecA protein of L. interrogans serovar hardjo, the RecA protein of the Leptospira serovars and L. illini were synthesized at elevated levels following treatment of cells with nalidixic acid. The level of detectable RecA correlated with previous studies demonstrating that free-living cells of L. biflexa serovars and L. illini were considerably more resistant to DNA-damaging agents than were those of parasitic L. interrogans serovars. RecA protein was not detected in cells of virulent Treponema pallidum or Borrelia burgdorferi.     

Regulation of the SOS response in Bacillus subtilis: evidence for a LexA repressor homolog.

The inducible SOS response for DNA repair and mutagenesis in the bacterium Bacillus subtilis resembles the extensively characterized SOS system of Escherichia coli. In this report, we demonstrate that the cellular repressor of the E. coli SOS system, the LexA protein, is specifically cleaved in B. subtilis following exposure of the cells to DNA-damaging treatments that induce the SOS response. The in vivo cleavage of LexA is dependent upon the functions of the E. coli RecA protein homolog in B. subtilis (B. subtilis RecA) and results in the same two cleavage fragments as produced in E. coli cells following the induction of the SOS response. We also show that a mutant form of the E. coli RecA protein (RecA430) can partially substitute for the nonfunctional cellular RecA protein in the B. subtilis recA4 mutant, in a manner consistent with its known activities and deficiencies in E. coli. RecA430 protein, which has impaired repressor cleaving (LexA, UmuD, and bacteriophage lambda cI) functions in E.coli, partially restores genetic exchange to B. subtilis recA4 strains but, unlike wild-type E. coli RecA protein, is not capable of inducing SOS functions (expression of DNA damage-inducible [din::Tn917-lacZ] operons or RecA synthesis) in B. subtilis in response to DNA-damaging agents or those functions that normally accompany the development of physiological competence. Our results provide support for the existence of a cellular repressor in B. subtilis that is functionally homologous to the E. coli LexA repressor and suggest that the mechanism by which B. subtilis RecA protein (like RecA of E. coli) becomes activated to promote the induction of the SOS response is also conserved.     

Evidence for RecA protein association with the cell membrane and for changes in the levels of major outer membrane proteins in SOS-induced Escherichia coli cells.

Membrane fractions from Escherichia coli cells expressing DNA damage-inducible (SOS) functions contain elevated quantities of RecA protein (L. J. Gudas and A. B. Pardee, J. Mol. Biol. 101:459-477, 1976). We used two-dimensional polyacrylamide gel electrophoresis to separate membrane proteins from several strains to determine whether this effect is an artifact due to contamination of membranes during preparation by the large amount of cytoplasmic RecA present in SOS-induced cells. We found that amplification of RecA+ protein without a DNA-damaging treatment does not result in increased RecA-membrane association, whether recA is depressed specifically by an operator-constitutive recA allele or coordinately with other SOS genes by a lexA mutation that inactivates their common repressor. In contrast, large amounts of RecA appear in membrane fractions from undamaged cells of an SOS-constitutive strain carrying recA730, which encodes a spontaneously SOS-activated RecA. We conclude that the increased association of RecA with the membrane fraction requires the presence of the activated form of RecA, and that this association may contribute significantly to the SOS response. We describe also striking effects of SOS expression on the levels of the outer membrane proteins OmpA, OmpC, and OmpF.     

Defective dissociation of a "slow" RecA mutant protein imparts an Escherichia coli growth defect

The RecA and some related proteins possess a simple motif, called (KR)X(KR), that (in RecA) consists of two lysine residues at positions 248 and 250 at the subunit-subunit interface. This study and previous work implicate this RecA motif in the following: (a) catalyzing ATP hydrolysis in trans,(b) coordinating the ATP hydrolytic cycles of adjacent subunits, (c) governing the rate of ATP hydrolysis, and (d) coupling the ATP hydrolysis to work (in this case DNA strand exchange). The conservative K250R mutation leaves RecA nucleoprotein filament formation largely intact. However, ATP hydrolysis is slowed to less than 15% of the wild-type rate. DNA strand exchange is also slowed commensurate with the rate of ATP hydrolysis. The results reinforce the idea of a tight coupling between ATP hydrolysis and DNA strand exchange. When a plasmid-borne RecA K250R protein is expressed in a cell otherwise lacking RecA protein, the growth of the cells is severely curtailed. The slow growth defect is alleviated in cells lacking RecFOR function, suggesting that the defect reflects loading of RecA at stalled replication forks. Suppressors occur as recA gene alterations, and their properties indicate that limited dissociation by RecA K250R confers the slow growth phenotype. Overall, the results suggest that recombinational DNA repair is a common occurrence in cells. RecA protein plays a sufficiently intimate role in the bacterial cell cycle that its properties can limit the growth rate of a bacterial culture.     

Reversible inhibition of protein splicing by zinc ion

Protein splicing involves the self-catalyzed excision of a protein-splicing element, the intein, from flanking polypeptides, the exteins, which are concomitantly joined by a peptide bond. Taking advantage of recently developed in vitro systems in which protein splicing occurs in trans to assay for protein-splicing inhibitors, we discovered that low concentrations of Zn(2+) inhibited splicing mediated both by the RecA intein from Mycobacterium tuberculosis and by the naturally split DnaE intein from Synechocystis sp. PCC6803. Inhibition by Zn(2+) was also observed with a cis-splicing system involving the RecA intein. In all experimental systems used, inhibition by Zn(2+) could be completely reversed by the addition of EDTA. Zinc ion also inhibited hydroxylamine-dependent N-terminal cleavage of the RecA intein. All other divalent transition metal ions tested were less effective as inhibitors than Zn(2+). The reversible inhibition by Zn(2+) should be useful in studies of the mechanism of protein splicing and allow structural studies of unmodified protein-splicing precursors.     

Immunological quantification of recA protein in cell extracts ofE. coli after exposure to chemical mutagens or UV radiation

Increased synthesis of RecA protein is induced inE. coli cells after their damage, the rate of synthesis being dependent on the extent of DNA alterations. The level of the RecA protein was determined inE. coli cell extracts after damage induced by NQO, MNNG, MMC, NAL or UV radiation, using competitive enzyme-linked immunosorbent assay (ELISA). PurifiedE. coli RecA protein and rabbit monospecific polyclonal antibodies against it were prepared for the quantitative assay. The level of theRecA protein was increased after treatment with all mutagens. Contrary to other induced proteins, the synthesis of the RecA protein increased within 30 min after damage with UV radiation at a relatively slow rate. The ELISA method made it possible to determine 0.5–50 ng of the RecA protein in bacterial extracts. The method can be employed as an auxiliary test for DNA damage determination and also in studied concerning the role of the RecA protein in repair processes. Translated by I. Miler