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PubMed1981

A temperature sensitive Reca protein of Escherichia coli.

The temperature sensitive allele recA200 has been cloned into the multiple copy number plasmid pBR322 and the gene product isolated. The purified RecA200 protein is temperature sensitive in ability to cleave the phage lambda and LexA repressors in vitro and also in ability to promote a successful search for homology between single stranded DNA and a homologous duplex leading to D-loop formation. However, at the non-permissive temperature the RecA200 protein has approximately wild type single stranded DNA dependent ATPase activity and ability to promote pairing between homologous single DNA strands. The demonstration that the temperature sensitivity in vivo can be correlated with the temperature sensitive cleavage of the lambda and LexA repressors in vitro and also with D-loop formation shows that these in vitro reactions, which require large amounts of RecA protein, are not carried out by trace amounts of contaminating proteins.

Bacterial Proteins↗
PubMed1981

Two mutations that alter the regulatory activity of E. coli recA protein.

The Escherichia coli recA gene product is both a direct participant and a central regulatory element in important processes of DNA repair, including one that is probably responsible for all radiation mutagenesis and some chemical mutagenesis. It has the direct function of catalysing pairing of single-stranded DNA to a homologous region in duplex DNA, a reaction thought to be fundamental to genetic recombination. This activity of recA protein probably contributes to DNA repair by promoting recombination between damaged DNA molecules. We have shown that recA protein also has a regulatory function, mediated by its ability to destroy repressors and possibly other proteins by proteolytic cleavage, leading to the induction of certain genes; these include recA itself, regulated by the lexA gene product, other genes probably involved in DNA repair, some of which are also regulated by the lexA gene product, and the early genes of temperate bacteriophages, which are regulated by their repressors, recA protein is activated to cleave repressors in vitro by interaction with ATP (or the ATP analogue ATP-gamma S) and a polynucleotide such as single-stranded DNA, probably the same interaction that initiates the strand-pairing reaction. We have proposed that recA protein is also activated to attack repressors in vivo when it interacts with single-stranded DNA; DNA-damaging treatments such as UV-ray irradiation would thereby invoke the recA-dependent functions of recombination, repair, mutagenesis and prophage induction. As further evidence that the proteolytic activity of recA protein is responsible for its regulatory function, we show here that the ability of two mutationally altered recA proteins to cleave phage lambda repressor correlates with the ability of the mutant cells to induce prophage.

Adenosine Triphosphatases↗
PubMed1997

The Rex phenotype of altruistic cell death following infection of a lambda lysogen by T4rII mutants is suppressed by plasmids expressing OOP RNA.

The coordinate expression from induced lambda prophages of pLit-rexB-tImm (late immunity transcription, LIT RNA) and po-oop-t(o) (OOP RNA) has remained unexplained. The initial assigned sequence for pLit bore no relationship to po. We have identified two promoter sites for independent rexB transcription, denoted here pLit2 and pLit1, which are separated by about 330 bp. The upstream pLit1 site shares with po a common 9 bp sequence between the -10 and -35 regions, with strong homology to aspects of the SOS box or LexA operator site. This sequence is also found within OOP RNA, suggesting that OOP RNA, or another regulatory factor recognizing the common sequence, was involved in the regulation of rexB expression and hence Rex exclusion. We measured the influence of OOP synthesis from plasmids on the Rex phenotype, finding that plasmids producing OOP can suppress Rex exclusion by a lambda prophage. The possibility was suggested that low level constitutive rexB transcription occurs from pLit2. Potential binding sites were identified for DnaA, for the LexA, CI and Cro repressors and for lambda O protein in the 80 nt DNA interval upstream from and including pLit1, suggesting a complex regulatory pattern for rexB expression from this promoter.

Bacteriophage lambda↗
PubMed1990

Regulation in repressor inactivation by RecA protein.

Treatments that damage DNA or inhibit DNA synthesis in E. coli induce the expression of a set of functions called SOS functions that are involved in DNA repair, mutagenesis, arrest of cell division and prophage induction. Induction of SOS functions is triggered by inactivation of the LexA repressor or a phage repressor. Inactivation of these repressors results from their cleavage by the E. coli RecA protein in the presence of single-stranded DNA and a nucleoside triphosphate. We found that these cleavage reactions are controlled by two mechanisms in vitro: one is through the structural change of the RecA protein in the ternary complex, RecA-ssDNA-ATP-gamma-S. The active ternary complex is formed by binding of ATP-gamma-S to a complex of RecA protein and ssDNA. On the other hand, when the RecA protein binds to ATP-gamma-S prior to its binding to ssDNA, the resulting complex has no or only very weak cleavage activity toward the repressor. This structural change is negatively controlled by its C-terminal part. The loss of the 25 amino acid residues from the C-terminal leads the RecA protein to stable binding to dsDNA as well as ssDNA, and the protein takes the activated form for the repressor cleavage constitutively. The other mechanism is through the structural change of the repressor. The cleavage reaction of a phi 80cI repressor is greatly stimulated by the presence of d(G-G), and d(G-G) stimulates the cleavage by binding to the C-terminal half of the phi 80cI repressor. Moreover, the C-terminal fragment of the cleaved products of the 80cI repressor was able to cleave a phi 80cI-lambda chimeric repressor. These results strongly suggested that the active site of the repressor cleavage was located in the C-terminal domain of the repressor and that the C-terminal fragment produced by the cleavage could cleave the repressor.

DNA Damage↗
PubMed1989

DNA repair mechanisms affecting cytotoxicity by streptozotocin in E. coli.

Mechanisms underlying cytotoxicity by the monofunctional nitrosourea streptozotocin (STZ) were evaluated in DNA repair-deficient E. coli mutants. Strains not proficient in recombinational repair which lack either RecA protein or RecBC gene products were highly sensitive to STZ. In contrast, cells that constitutively synthesize RecA protein and cannot initiate SOS repair mechanisms because of uncleavable LexA repressor (recAo98 lexA3) were resistant to this drug compared to a lexA3 strain. Further, E. coli cells lacking both 3-methyladenine DNA glycosylases I (tag) and II (alkA) also were highly sensitive to STZ. DNA synthesis was most inhibited by STZ in recA and alkA tag E. coli mutants, but was suppressed less markedly in wild-type and recBC cells. DNA degradation was most extensive in recA E. coli after STZ treatment, while comparable in recBC, alkA tag, and wild-type cells. Although increased single-stranded DNA breaks were present after STZ treatment in recA and recBC mutants compared to the wild type, no significant increase in DNA single-stranded breaks was noted in alkA tag E. coli. Further, DNA breaks in recBC cells were repaired, while those present in recA cells were not. These findings establish the critical importance of both recombinational repair and 3-methyladenine DNA glycosylase in ameliorating cytotoxic effects and DNA damage caused by STZ in E. coli.

Bacterial Proteins↗
PubMed1986

New role for photoreversible pyrimidine dimers in induction of prototrophic mutations in excision-deficient Escherichia coli by UV light.

UV mutagenesis to His+ in certain recA441 lexA51 bacteria was not photoreversible, indicating that pyrimidine dimers are not target lesions. Photoreversibility was observed in recA+ lexA51 bacteria, showing that pyrimidine dimers are needed to activate the recA+ protein (unlike the recA441 protein) to perform a function in UV mutagenesis distinct from cleavage of the lexA repressor.

DNA Repair↗
PubMed1989

[Interference of inducible repair processes in Escherichia coli].

Radiation and the majority of chemical mutagens produce lesions in the DNA of cells which provoke the induction--as a reverse response--of some inducible repair processes. One of them is the adaptive response--highly specific in the repair of damages, induced by alkylating agents. This repair pathway decreases the toxic and mutagenic effects of many alkylating agents and can be induced in Escherichia coli cells exposed to sublethal concentrations of the same agents. By contrast, the SOS repair pathway in E. coli is non-specific and transient phenomenon which leads, among other things, to bacterial mutagenesis. It is controlled by the regulatory RecA protein which in its activated form promotes the cleavage of LexA repressor, allowing for the increased transcription of about 17 repressed genes--SOS regulon. The latter is not associated with the adaptive response. Nevertheless, there are experimental data indicating that the adaptive response is able to reduce some functions of the SOS repair activity--W-reactivation, W-mutagenesis and lambda phage induction. A relatively new bacterial short-term assay for genotoxicity, the SOS chromotest with E. coli PQ37 as an indicator organism, makes it possible to measure SOS induction indirectly, on the basis of a simple colorimetric assay. In the present study, the SOS chromotest in a completely automated system "Bioscreen C" was used to study interference between the adaptive and SOS responses in E. coli. Our data indicate that there is an inhibitory effect of the adaptive response on the SOS induction, as well as the negative interference between two successive SOS responses, at a transcriptional level of the SOS induction.

Adaptation, Biological↗
PubMedDate not supplied

recA operator mutations and their usefulness.

Mutations at three positions in the operator for recA have been detected, cloned and sequenced. Derepressed amounts of recA protein vary over a forty-fold range and correlate well with reduced affinities for lexA repressor of the mutant operators. One mutant confirms the region of major groove interaction between repressor and operator. Another has been used to demonstrate that RecF pathway genes other than recA are under lexA control.

Bacterial Proteins↗
PubMed1984

Cloning and characterization of recA genes froM Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r.

The recA genes of Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r have been isolated and introduced into Escherichia coli K-12. All the heterologous genes restore resistance to killing by UV irradiation and the mutagen 4-nitroquinoline-1-oxide in RecA- E. coli K-12 hosts. Recombination proficiency is also restored as measured by formation of Lac+ recombinants from duplicated mutant lacZ genes and the ability to propagate phage lambda derivatives requiring host recombination functions for growth (Fec-). The cloned heterologous genes increase the spontaneous induction of lambda prophage in lysogens of a recA strain. Addition of mitomycin C stimulates phage production in cells carrying the E. coli B/r and S. flexneri recA genes, but little or no stimulation is seen in cells carrying the E. carotovora and P. vulgaris recA genes. After treatment with nalidixic acid, the heterologous RecA proteins are synthesized at elevated levels, a result consistent with their regulation by the E. coli K-12 LexA repressor. Southern hybridization and preliminary restriction analysis indicate divergence among the coding sequences, but antibodies prepared against the E. coli K-12 RecA protein cross-react with the heterologous enzymes, indicating structural conservation among these proteins.

Bacteria↗
PubMed1983

Evolution of cellular ATP concentration after UV-mediated induction of SOS system in Escherichia coli.

UV-irradiation of E. coli induces a two fold increase in ATP pool in the first 20 min. Afterwards, in RecA+ strains ATP level drops quickly below values of non irradiated cells. Mutants of E. coli defective in RecA protein or with either RecA protease activity deficient or protease resistant LexA repressor do not present this decrease, showing that it is due to cleavage of LexA repressor by RecA protease. The ATP increase produced in the first 20 min is dependent on RecBC exonuclease activity and it must be due to substrate level phosphorylation since an uncoupler such as dinitrophenol does not affect it.

Adenosine Triphosphate↗
PubMed1989

Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia coli.

The nucleotide sequence of the recA gene of Thiobacillus ferrooxidans has been determined. No SOS box characteristic of LexA-regulated promoters could be identified in the 196-bp region upstream from the coding region. The cloned T. ferrooxidans recA gene was expressed in Escherichia coli from both the lambda pR and lac promoters. It was not expressed from the 2.2-kb of T. ferrooxidans DNA preceding the gene. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and Pseudomonas aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with proteolytic activity have been substituted, the cloned protein has retained protease activity towards the lambda and E. coli LexA repressors.

Amino Acid Sequence↗
PubMed1985

Characterization of DNA polymerase I*, a form of DNA polymerase I found in Escherichia coli expressing SOS functions.

DNA polymerase I* is a form of the DNA polymerase I isolated from Escherichia coli which are expressing recA/lexA (SOS) functions. Induction of recA or polA1 cells by nalidixic acid does not result in the appearance of pol I*, but lexA or recA mutants that are constitutive for SOS functions constitutively express pol I* and mutants which lack functional recA protein produce pol I* when they carry a lexA mutation which renders the lexA repressor inoperative. Pol I* has been induced by nalidixic acid in dinA, dinD, dinF, and umuC mutants. Polymerase I* has a lower affinity for single-stranded DNA-agarose than polymerase I and it sediments through sucrose gradients in a dispersed manner between 6.6-10.5 S, whereas polymerase I sediments at 5 S. Whereas pol I* migrates significantly faster than pol I in nondenaturing polyacrylamide gels, the active polypeptide of both forms migrates at the same rate in denaturing polyacrylamide gels. Compared with polymerase I, polymerase I* has an enhanced capacity to incorporate the adenine analog, 2-amino-purine, into activated salmon sperm DNA and a relatively low fidelity in replicating synthetic polydeoxyribonucleotides. Both the 3'----5' (proofreading) and 5'----3' (nick-translational) exonuclease activities of pol I* and pol I are indistinguishable. Estimates of processivity give a value of approximately 6 for both forms of the enzyme.

Centrifugation, Density Gradient↗
PubMed1982

Cell survival, UV-reactivation and induction of prophage lambda in Escherichia coli K12 overproducing RecA protein.

The effect of the cellular level of RecA protein on the ability of E. coli K12 bacteria to (i) survive UV-irradiation (ii) promote UV-reactivation of UV-damaged phage lambda (iii) induce prophage lambda was determined in bacterial mutants with discrete increasing levels of RecA protein. The various levels of RecA protein were obtained by combining lexA and recA alleles. Except for the double mutant lexA3 recAo98, whose repair ability was 25% less than that observed in wild type bacteria, bacterial survival was proportional to the level of RecA protein measured after 90 min of incubation. In lexA3 recAo98 bacteria, RecA protein, at a constitutive high basal level, failed to compensate totally for the lack of LexA repressor cleavage; UV-reactivation of UV-damaged phage lambda was not restored; yet, prophage lambda was induced with 35% efficiency. Efficient UV-induction of prophage lambda is linked to the induction of lexA-controlled host processes that repair the UV-damaged prophage.

Bacterial Proteins↗
PubMed1988

Cleavage of bacteriophage phi 80 CI repressor by RecA protein.

We have purified the CI repressor protein of bacteriophage phi 80. Its N-terminal amino acid sequence and its amino acid composition agree with those predicted from the nucleotide sequence of the cI gene. The phi 80 CI repressor was cleaved at a Cys-Gly bond by the wildtype RecA protein in the presence of single-stranded DNA and ATP or its analogues. This cleavage site is different from other repressors such as LexA, lambda CI and P22 C2, which were cleaved at an Ala-Gly bond. The phi 80 CI repressor was cleaved at the same site by the RecA430 protein, but was not cleaved by the RecA1 protein. This effect of the bacterial recA mutations on cleavage is consistent with the fact that prophage phi 80 in recA430 cells can be induced by irradiation with ultraviolet light, while the prophage in recA1 cells cannot.

Amino Acid Sequence↗
PubMed1997

Identification of the Saccharomyces cerevisiae genes STB1-STB5 encoding Sin3p binding proteins.

The yeast SIN3 gene functions as a transcriptional repressor, despite the fact that Sin3p does not bind DNA directly. We have conducted a two-hybrid screen to look for proteins that interact with Sin3p, using the PAH2 domain of Sin3p as bait. Five new genes, STB1-STB5 were identified, as well as the STB6 gene, which is similar to STB2. STB1, STB2, STB3, and STB6 are novel genes, and STB4 and STB5 encode C6 zinc cluster DNA-binding proteins. None of these genes is essential for viability, and several of these genes may encode transcriptional activators. Several special problems were encountered in using a transcriptional repressor in a two-hybrid screen. For example, the STB genes will interact with a LexA-Sin3(PAH2) fusion protein containing a region of Sin3p, but a LexA-Sin3p fusion protein containing full-length Sin3p, along with a STB clone, does not produce two-hybrid activation of a transcriptional reporter. In addition, a sin3 mutation reduces the transcriptional activation by two-hybrid partners, suggesting that a sin3 mutation reduces the transcriptional efficiency of the Gal4p and VP16 activation domains. We have shown previously that Sin3p is part of a large multiprotein complex, and we show here that Stb1p and Stb2p are present in this complex.

Base Sequence↗
PubMed1981

Organization of the lexA gene of Escherichia coli and nucleotide sequence of the regulatory region.

The product of the lexA gene of Escherichia coli has been shown to regulate expression of the several cellular functions (SOS functions) induced by treatments which abruptly inhibit DNA synthesis. We have cloned and mapped the lexA gene on a small segment of approximately 600 base pairs. The lexA promotor was located by transcription R-loop analysis, and the lexA product of 22,000 daltons was identified by protein synthesis in vitro. An unknown gene was found which directed the synthesis of a protein of 35,000 daltons in a region downstream from the lexA gene. Nucleotide sequence of the regulatory region of the lexA gene was determined. The sequence contained inverted repeats homologous to that of the recA regulatory region. These inverted repeats may be recognized by the lexA protein, because the protein is considered to repress both the genes as a common repressor.

Bacterial Proteins↗
PubMed1982

Mutagenesis and cellular responses to DNA damage.

Treatment of Escherichia coli with DNA-damaging agents results in the increased expression of a set of din (damage-inducible) genes. We have studied the regulation and function of these genes by using the Mud(Ap, lac) bacteriophage to obtain fusions of the beta-galactosidase structural gene to the promoters of various din genes. By this technique, we have shown that the uvrA, uvrB, and umuC genes are induced by UV and other DNA damaging agents. The products of the uvrA and uvrB genes are required for the excision repair of pyrimidine dimers and other bulky lesions; the umuC gene product is required for most chemical mutagenesis in E. coli. Genetic analyses of all of the din-lac fusions isolated to date indicate that lexA is the direct repressor of each of the din genes and that proteolytic cleavage of the lexA protein is required for their induction. We have also been studying the mechanism by which the clinically isolated plasmid pKM101 increases the susceptibility of cells to chemical mutagenesis. Inasmuch as the effects of pKM101 on mutagenesis are recA+ lexA+ -dependent and the plasmid can suppress the nonmutability of a umuC mutant, it seems likely that pKM101 may carry an analog of the chromosomal umuC gene. By insertion mutagenesis using Tn5, we identified an approximately 2,000-base pair region of pKM101 which is necessary for its effects on mutagenesis.

Bacterial Proteins↗
PubMed1988

Transcriptional and translational analyses of recA mutant alleles in Pseudomonas aeruginosa.

Recombinant plasmids containing the recA gene from Pseudomonas aeruginosa were used in complementation, transcriptional, and translational studies to examine the nature of rec-102 and rec-2, mutations which confer a recA-like mutant phenotype on P. aeruginosa PAO strains. For comparison, recA7::Tn501 mutants of strain PAO were constructed by gene replacement. The rec-2 and rec-102 alleles were shown to be recA alleles; plasmids containing the recA gene complemented the three rec mutant strains for defects associated with recA mutation. Northern blot analyses indicated that the recA gene in P. aeruginosa was transcribed as two distinct mRNAs of approximately 1.2 and 1.4 kilobases (kb). A plasmid encoding both transcripts of recA complemented all defects associated with the three recA mutations rec-2, rec-102, and recA7. However, a 2.4-kb subclone (pJH13) encoding only the smaller transcript of the recA gene was expressed differently in the three recA allele backgrounds and served as a tool to distinguish the nature of the rec-2 and rec-102 mutations in recA. A minicell analysis showed that a plasmid expressing both of the recA gene transcripts or one that expressed only the smaller transcript both produced the same 42-kilodalton recA protein. A chloramphenicol acetyltransferase gene fusion in the 3' end of the recA transcript showed that the recA gene of P. aeruginosa was induced following treatment with a DNA-damaging agent (methyl methanesulfonate). The recA7 mutant constructed here showed no recA-related transcript or protein under inducing conditions, and pJH13 in this host produced only low levels of the smaller recA transcript and low levels of recA protein. The rec-2 mutant produced a detectable transcript but no recA protein following induction. The presence of low levels of activated recA protein encoded by pJH13 in the rec-2 mutant resulted in wild-type transcriptional levels of chromosomally encoded recA, but no recA protein was detectable. Thus, the rec-2 allele of recA was normal with respect to induction of mRNA, but these transcripts were defective in either translation or synthesis of a stable protein. The rec-102 mutant also produced a detectable transcript and no recA protein following induction, but having pJH13 in the cell to produce low levels of activated recA protein resulted in overproduction of chromosomally encoded recA transcripts and active recA protein. Thus, the recA defect in the rec-102 mutant is apparently in the interaction between recA and a lexA-like repressor.

Alleles↗