Therefore, what are recombination proteins there for?

The order of discovery can have a profound effect upon the way in which we think about the function of a gene. In E. coli, recA is nearly essential for cell survival in the presence of DNA damage. However, recA was originally identified, as a gene required to obtain recombinant DNA molecules in conjugating bacteria. As a result, it has been frequently assumed that recA promotes the survival of bacteria containing DNA damage by recombination in which DNA strand exchanges occur. We now know that several of the processes that interact with or are controlled by recA, such as excision repair and translesion synthesis, operate to ensure that DNA replication occurs processively without strand exchanges. Yet the view persists in the literature that recA functions primarily to promote recombination during DNA repair. With the benefit of hindsight and more than three decades of additional research, we reexamine some of the classical experiments that established the concept of DNA repair by recombination, and we consider the possibilities that recombination is not an efficient mechanism for rescuing damaged cells, and that recA may be important for maintaining processive replication in a manner that does not generally promote recombination.

Pseudomonas aeruginosa exoenzyme S disrupts Ras-mediated signal transduction by inhibiting guanine nucleotide exchange factor-catalyzed nucleotide exchange.

Pseudomonas aeruginosa exoenzyme S double ADP-ribosylates Ras at Arg(41) and Arg(128). Since Arg(41) is adjacent to the switch 1 region of Ras, ADP-ribosylation could interfere with Ras-mediated signal transduction via several mechanisms, including interaction with Raf, or guanine nucleotide exchange factor-stimulated or intrinsic nucleotide exchange. Initial experiments showed that ADP-ribosylated Ras (ADP-r-Ras) and unmodified Ras (Ras) interacted with Raf with equal efficiencies, indicating that ADP-ribosylation did not interfere with Ras-Raf interactions. While ADP-r-Ras and Ras possessed equivalent intrinsic nucleotide exchange rates, guanine nucleotide exchange factor (Cdc25) stimulated the nucleotide exchange of ADP-r-Ras at a 3-fold slower rate than Ras. ADP-r-Ras did not affect the nucleotide exchange of Ras, indicating that the ADP-ribosylation of Ras was not a dominant negative phenotype. Ras-R41K and ADP-r-Ras R41K possessed similar exchange rates as Ras, indicating that ADP-ribosylation at Arg(128) did not inhibit Cdc25-stimulated nucleotide exchange. Consistent with the slower nucleotide exchange rate of ADP-r-Ras as compared with Ras, ADP-r-Ras bound its guanine nucleotide exchange factor (Cdc25) less efficiently than Ras in direct binding experiments. Together, these data indicate that ADP-ribosylation of Ras at Arg(41) disrupts Ras-Cdc25 interactions, which inhibits the rate-limiting step in Ras signal transduction, the activation of Ras by its guanine nucleotide exchange factor.

Expression and nucleotide excision repair of a UV-irradiated reporter gene in unirradiated human cells.

It has been suggested that reactivation of damaged reporter genes introduced into cultured mammalian cells reflects transcription-coupled nucleotide excision repair. To evaluate this possibility directly, we introduced a UV-irradiated shuttle vector, pCMV beta, into unirradiated human cells and compared expression of the reporter gene (lacZ) with repair of cyclobutane pyrimidine dimers (CPDs). Expression of the irradiated reporter gene was more UV resistant in XPC cells, which are deficient in global genome repair, than in CSB cells, which are deficient in transcription-coupled repair. These results are consistent with the idea that repair of the reporter gene is primarily dependent upon transcription-coupled repair. However, when the plasmid DNA was analyzed for removal of CPDs, no clear evidence was obtained for transcription-coupled repair either in XPC cells or in cells with normal repair capacity.

Pseudomonas aeruginosa exoenzyme S, a double ADP-ribosyltransferase, resembles vertebrate mono-ADP-ribosyltransferases.

Previous data indicated that Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras at multiple sites. One site appeared to be Arg41, but the second site could not be localized. In this study, the sites of ADP-ribosylation of c-Ha-Ras by ExoS were directly determined. Under saturating conditions, ExoS ADP-ribosylated Ras to a stoichiometry of 2 mol of ADP-ribose incorporated per mol of Ras. Nucleotide occupancy did not influence the stoichiometry or velocity of ADP-ribosylation of Ras by ExoS. Edman degradation and mass spectrometry of V8 protease generated peptides of ADP-ribosylated Ras identified the sites of ADP-ribosylation to be Arg41 and Arg128. ExoS ADP-ribosylated the double mutant, RasR41K,R128K, to a stoichiometry of 1 mol of ADP-ribose incorporated per mol of Ras, which indicated that Ras possessed an alternative site of ADP-ribosylation. The alternative site of ADP-ribosylation on Ras was identified as Arg135, which was on the same alpha-helix as Arg128. Arg41 and Arg128 are located within two different secondary structure motifs, beta-sheet and alpha-helix, respectively, and are spatially separated within the three-dimensional structure of Ras. The fact that ExoS could ADP-ribosylate a target protein at multiple sites, along with earlier observations that ExoS could ADP-ribosylate numerous target proteins, were properties that have been attributed to several vertebrate ADP-ribosyltransferases. This prompted a detailed alignment study which showed that the catalytic domain of ExoS possessed considerably more primary amino acid homology with the vertebrate mono-ADP-ribosyltransferases than the bacterial ADP-ribosyltransferases. These data are consistent with the hypothesis that ExoS may represent an evolutionary link between bacterial and vertebrate mono-ADP-ribosyltransferases.

Pseudomonas aeruginosa exoenzyme S ADP-ribosylates Ras at multiple sites.

Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras to a stoichiometry of approximately 2 molecules of ADP-ribose incorporated per molecule of Ras, which suggested that ExoS could ADP-ribosylate Ras at more than one arginine residue. SDS-polyacrylamide gel electrophoresis analysis showed that ADP-ribosylated Ras possessed a slower mobility than non-ADP-ribosylated Ras. Analysis of the ADP-ribosylation of in vitro transcribed/translated Ras by ExoS identified two electrophoretically shifted forms of Ras, which was consistent with the ADP-ribosylation of Ras at two distinct arginine residues. Analysis of ADP-ribosylated in vitro transcribed/translated Ras mutants possessing individual Arg-to-Ala substitutions showed that Arg-41 was the preferred site of ADP-ribosylation and that the second ADP-ribosylation event occurred at a slower rate than the ADP-ribosylation at Arg-41, but did not occur at a specific arginine residue. Analysis of bacterially expressed wild-type RasDeltaCAAX and RasDeltaCAAXR41K supported the conclusion that Arg-41 was the preferred site of ADP-ribosylation. Arg-41 is located adjacent to the switch 1 region of Ras, which is involved in effector interactions. Introduction of ExoS into eukaryotic cells inhibited Ras-mediated eukaryotic signal transduction since infection of PC-12 cells with an ExoS-producing strain of P. aeruginosa inhibited nerve growth factor-stimulated neurite formation. This is the first demonstration that ExoS disrupts a Ras-mediated signal transduction pathway.

Two modes of ligand binding in maltose-binding protein of Escherichia coli. Functional significance in active transport.

In the preceding two papers (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609; Hall, J. A., Thorgeirson, T. E., Liu, J., Shin, Y.-E., and Nikaido, H. (1997) J. Biol. Chem. 272, 17610-17614), we showed that ligands that bind to the Escherichia coli maltose-binding protein (MBP) without producing the closure of its two lobes are not transported into the cytoplasm. Here, we examine various combinations of ligands, MBPs, and membrane-associated transporters, by utilizing reconstituted proteoliposomes, right side-out membrane vesicles, and intact cells. Closed forms of wild type MBP, complexed with maltose or maltodextrins, interacted with wild type transporter complex to stimulate the hydrolysis of ATP by MalK ATPase located on the other side of the membrane, as shown earlier for the maltose-MBP complex (Davidson, A. L., Shuman, H. A., and Nikaido, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2360-2364). In contrast, open forms of liganded MBPs, such as the complex containing wild type MBP and reduced, oxidized, or cyclic maltodextrins or the complex containing the mutant MBP MalE254 and unmodified maltodextrins, did not stimulate ATP hydrolysis, suggesting that the proper interaction between the ligand-MBP complex and the external surface of the transporter requires the former to be in the closed conformation. However, when a mutant transporter containing MalG511 was used, the already significant basal level of ATP hydrolysis was further stimulated not only by ligand MBPs in the closed form but also by those in the open form (except that containing beta-cyclodextrin), data suggesting that the mutant transporter does not always require the closed MBP complex presumably because of its exceptionally strong affinity to MBP, described earlier (Dean, D. A., Hor, L.-I., Shuman, H. A., and Nikaido, H. (1992) Mol. Microbiol. 6, 2033-2040). Furthermore, this mutant transporter was able to transport reduced maltodextrin, and cells expressing the transporter were able to grow by using reduced maltodextrin, if the periplasmic concentrations of MBP were kept low so as not to inhibit the transport process.

Removal of cyclobutane pyrimidine dimers from a UV-irradiated shuttle vector introduced into human cells.

A shuttle vector (pZH-1) carrying the E. coli lacZ gene under control of the SV40 early promoter was irradiated with UV and introduced into repair-proficient or repair-deficient human cell lines. The expression of irradiated lacZ compared to unirradiated lacZ was greater in repair-proficient cells (HT-1080) than in repair-deficient cells (XP12RO-SV40) belonging to xeroderma pigmentosum complementation group A. To ascertain whether the expression of lacZ in the repair-proficient cells was correlated with the removal of cyclobutane pyrimidine dimers (CPDs), we purified DNA from the recipient cells and used the CPD-specific enzyme T4 endonuclease V to measure the frequency of CPDs remaining in the plasmid as a whole and in two restriction fragments derived from it. We found that removal of CPDs occurred in both fragments in the repair-proficient cells but not in the repair-deficient cells. Our results provide the first direct evidence for the removal of CPDs from UV irradiated plasmids introduced into human cells and support the notion that expression of the UV-damaged lacZ gene in repair-proficient human cells reflects the removal of transcription blocking lesions from the gene.

Temperature dependent survival of UV-irradiated Escherichia coli K12.

We have found that several excision deficient derivatives of Escherichia coli K12 survive better after UV irradiation if incubated at 42 degrees C than if incubated at 30 degrees C. The highest survival was observed when incubation at 42 degrees C followed UV irradiation and was maintained for at least 16 h. Our results indicate that this temperature dependent resistance (TDR) requires a functional recA gene, but not uvrA, uvrB, recF, or recB genes, or the recA441 (tif-1) mutation which allows thermoinduction of the recA-lexA regulon. Our data are consistent with the idea that the increase in survival observed at 42 degrees C reflects enhanced daughter-strand gap repair by DNA strand exchange. Although the conditions used to elicit TDR can induce heat shock proteins and thermotolerance in E. coli, the relationship between the two responses remains to be elucidated.

Enhanced transforming activity of pSV2 plasmids in human cells depends upon the type of damage introduced into the plasmid.

When pSV2-gpt or pSV2-neo plasmids are introduced into human cells by calcium phosphate coprecipitation, the yield of stable transformants (Gpt+ or Neo+) is increased by irradiating the respective plasmid DNA in vitro with UV (254 nm). To identify specific lesions that can increase the transforming activity of plasmids in human cells we examined pSV2 plasmids containing different types of damage. Of the lesions tested, cyclobutane pyrimidine dimers produced the greatest increase, and can nearly fully account for the effect of 254 nm UV on transformation. The enhancement of transformation produced by UV was not altered by the additional treatment of the plasmid DNA with T4 endonuclease V, an enzyme that nicks DNA specifically at pyrimidine dimers. Treatment of plasmid DNA with osmium tetroxide to produce thymine glycols, or with acid and heat to produce apurinic sites did not affect transformation frequency. The enhancement occurred in all the human cell lines tested, whether they contained or not sequences homologous to those in the plasmids, and was independent of the repair capacity of the recipient cells.

Enhanced transforming activity of ultraviolet-irradiated pSV2-gpt is due to damage outside the gpt transcription unit.

We have shown that when pSV2-gpt is introduced into human cells by calcium phosphate coprecipitation, the yield of Gpt+ transformants is increased by irradiating the plasmid with 254 nm uv. To elucidate the mechanism underlying this response, we constructed pSV2-gpt molecules in which the uv damage was confined to a particular region: a 3.0-kb region containing the pBR322 sequences and simian virus 40 (SV40) sequences not required for expression of the gpt gene, or a 2.3-kb fragment containing the Escherichia coli gpt gene together with the SV40 early promoter and sequences needed for splicing and polyadenylation. The transforming activity of the plasmid was greatly enhanced by uv damage confined to the 3.0-kb pBR322 region and increased linearly with uv dose up to 1 kJ/m2, but remained relatively constant at doses between 2 and 8 kJ/m2. Positioning the damaged region upstream, or both upstream and downstream, from the gpt transcription unit increased the uv enhancement slightly compared to positioning the damaged region only downstream. In contrast, transforming activity was significantly decreased by damage in the 2.3-kb gpt transcription unit. These results suggest that uv damage outside a selectable marker gene in a plasmid can increase the probability of stable integration of the plasmid into the genome of recipient cells without inhibiting expression of of the gene.

Processivity of T4 endonuclease V is sensitive to NaCl concentration.

We previously reported that endonuclease V of bacteriophage T4 reacts processively with pyrimidine dimers in UV-irradiated DNA, tending to react with all of the dimers on one DNA molecule before reacting with any dimers on another DNA molecule [Lloyd, R. S., Hanawalt, P. C., & Dodson, M. L. (1980) Nucleic Acids Res. 8, 5113-5127]. In this paper we show that this processivity depends upon salt concentration: it can be detected in 10 mM NaCl but not, by our methods, in 100 mM NaCl. In addition, we show that endonuclease V binds to unirradiated DNA in 10 mM NaCl but not in 100 mM NaCl. We conclude that T4 endonuclease V binds to pyrimidine dimers in a two-step process in 10 mM NaCl. It first binds electrostatically to undamaged sections of DNA, and it remains bound during the second step in which it "searches" for pyrimidine dimers. Our conclusion is analogous to the expanded target theory developed for Lac repressor [Berg, O. G., Winter, R. B., & von Hippel, P. H. (1981) Biochemistry 20, 6929-6948].

Differential expression of SOS genes in an E. coli mutant producing unstable lexA protein enhances excision repair but inhibits mutagenesis.

The lexA41 mutant of E. coli is a UV-resistant derivative of another mutant, lexA3, which produces a repressor that is not cleaved following inducing treatments. lexA41 carried an additional mutation which changed amino acid 132 in the LexA protein from Ala to Thr. The resultant protein was unstable and was degraded both before and after an inducing treatment. This instability was greater at 42 degrees than at 30 degrees. The protein was more stable in Lon- mutants at both temperatures. lac operon fusions to most of the genes in the SOS regulon were used to show that the various damage-inducible genes were derepressed to different extents. uvrA, B, and D were almost fully derepressed. Consistant with this finding, the rate of removal of T4 endonuclease V-sensitive sites was more rapid in the UV-irradiated lexA41 mutant than in normal cells, suggesting a more active excision repair system. We propose that the instability of the LexA41 protein reduces the intracellular concentration of repressor to a level that allows a high level of excision repair. The additional observation that SOS mutagenesis was only weakly induced in a lexA41 uvrA- mutant implies that the mutant protein partially represses one or more genes whose products promote SOS mutagenesis.

Effect of a lexA41(Ts) mutation on DNA repair in recA(Def) derivatives of Escherichia coli K-12.

Derivatives of Escherichia coli K-12 carrying a deletion of the recA gene survive exposure to UV (254 nm) better if they also contain the lexA41 mutation which codes for a labile LexA protein. This effect of the lexA41 mutation is not observed in comparable strains carrying a uvr A6 mutation. Using two independent methods to detect pyrimidine dimers we found that UV irradiated RecA deficient cells removed dimers from their DNA more rapidly if they contained the lexA41 mutation than if they contained the wild-type lexA gene. Our results are consistent with the idea that a relatively high level of UvrABC incision nuclease resulting from inefficient repression of the corresponding genes by the labile LexA41 protein facilitates excision of pyrimidine dimers from the DNA of UV irradiated cells.

Enhanced transformation of human cells by UV-irradiated pSV2 plasmids.

Irradiating the plasmid pSV2-gpt with UV (254 nm) doses up to 200 J m-2 caused a dose-dependent increase in the yield of Gpt+ transformants when the plasmid was introduced into human cells by calcium phosphate coprecipitation. UV doses greater than 1 kJ m-2 were required to reduce the efficiency of transformation below that obtained with unirradiated DNA.

den V gene of bacteriophage T4 determines a DNA glycosylase specific for pyrimidine dimers in DNA.

Endonuclease V of bacteriophage T4 has been described as an enzyme, coded for by the denV gene, that incises UV-irradiated DNA. It has recently been proposed that incision of irradiated DNA by this enzyme and the analogous "correndonucleases" I and II of Micrococcus luteus requires the sequential action of a pyrimidine dimer-specific DNA glycosylase and an apyrimidinic/apurinic endonuclease. In support of this two-step mechanism, we found that our preparations of T4 endonuclease V contained a DNA glycosylase activity that produced alkali-labile sites in irradiated DNA and an apyrimidinic/apurinic endonuclease activity that converted these sites to nicks. Both activities could be detected in the presence of 10 mM EDTA. In experiments designed to determine which of the activities is coded by the denV gene, we found that the glycosylase was more heat labile in extracts of Escherichia coli infected with either of two thermosensitive denV mutants than in extracts of cells infected with wild-type T4. In contrast, apyrimidinic/apurinic endonuclease activity was no more heat labile in extracts of the former than in extracts of the latter. Our results indicate that the denV gene codes for a DNA glycosylase specific for pyrimidine dimers.

Binding of T4 endonuclease V to deoxyribonucleic acid irradiated with ultraviolet light.

Endonuclease V of bacteriophage T4 binds to UV-irradiated deoxyribonucleic acid (DNA) but not to unirradiated DNA. We have developed an assay to detect this binding, based on the retention of enzyme--DNA complexes on nitrocellulose filters. The amount of complex retained, ascertained by using radioactive DNA, is a measure of T4 endonuclease V activity. The assay is simple, rapid, and specific, which makes it useful for detecting T4 endonuclease V activity both in crude lysates and in purified preparations. We have used it to monitor enzyme activity during purification and to study binding of the enzyme to DNA under conditions that minimize the ability of the enzyme to nick DNA. From our data we conclude that (1) T4 endonuclease V binds to UV-irradiated DNA but not to DNA that has been previously incised by the endonuclease, (2) equilibrium between the free and complexed form of the enzyme is attained under our reaction conditions, (3) dissociation of enzyme--DNA complexes is retarded by sodium cyanide, and (4) retention of enzyme--DNA complexes on nitrocellulose filters is enhanced by high concentrations of saline--citrate.