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PubMed1985

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.

Bacterial Proteins↗
PubMedDate not supplied

The two-step model of bacterial UV mutagenesis.

Recent results are discussed which have led to a two-step model for UV mutagenesis in excision-deficient Escherichia coli. After exposure to UV, the replication fork is assumed to continue until immediately before certain photoproducts where it stops and leaves a gap which cannot be dealt with by recombination repair. In the first (misincorporation) step, bases (a proportion of which are 'wrong') are postulated to be inserted opposite the photoproduct under the direct influence of the recA gene product. These misincorporated bases can be revealed as mutations by delayed photoreversal in umuD,C and lexA (ind-) bacteria. Their level is determined by the particular allele of recA that is present (recA441 greater than recA+ greater than recA430) and their rate of formation by the amount of recA protein in the cell and the degree of enrichment of the medium. No other protein needs to be synthesized for this step to occur. The second (bypass) step requires induced levels of the products of the umuD and C genes which are postulated to facilitate continued DNA synthesis on the priming end opposite the photoproduct. In principle, further errors could be made at this stage which might appear as 'hitch-hiking' rather than 'targeted' mutations.

Bacteria↗
PubMed1985

Isolation and analysis of a mutant of Escherichia coli hyper-resistant to near-ultraviolet light plus 8-methoxypsoralen.

A mutant of Escherichia coli K12 was isolated which shows enhanced resistance towards near-ultraviolet (NUV) light plus 8-methoxypsoralen (MPS) compared with its wild-type parent strain. The PUVA (NUV + MPS)-resistant strain remains as sensitive for far-ultraviolet (FUV) light as its parent strain. A recA- derivative of this mutant strain was as sensitive to PUVA as its reca- parental strain. A polyacrylamide gel electrophoresis study of total cell lysates from the mutant bacteria showed that a protein of approximately 55 kd was synthesised in higher concentrations compared with its synthesis in the wild-type parent strain. Furthermore, synthesis of this protein was reduced in the recA- derivative of the mutant strain suggesting that the recA gene product might be acting as a regulator of the synthesis of the 55-kd protein. It is suggested that in E. coli damage to DNA by PUVA can be repaired by a specific RecA LexA-inducible repair system and the repair efficiency is enhanced if the 55-kd protein is present in concentrations higher than that synthesised by the wild-type parent E. coli.

Bacterial Proteins↗
PubMed1984

Plasmid vectors designed for high-efficiency expression controlled by the portable recA promoter-operator of Escherichia coli.

A novel expression vector using the 236-bp promoter-operator fragment of the recA gene (recApo) of Escherichia coli has been constructed. This DNA fragment contains complete signals for the initiation of RNA synthesis, as well as for regulation by the lexA product, but lacks the coding sequence for the RecA protein. The strength of the recA promoter was examined by assaying beta-galactosidase activity expressed from a cro-lacZ fused gene placed downstream of the promoter. Under noninducing conditions, the promoter was regulated by the LexA protein, and the fused gene was expressed only weakly. Upon induction by nalidixic acid in a recA+ strain, high expression was observed for an extended period. After 5 h under inducing conditions, as much as 11% of the total cellular protein was cro-lacZ product. The expression level was higher than that from promoters of lac, trp, and lambda early genes.

Bacterial Proteins↗
PubMed2006

Genetic analysis of the anti-mutagenic effect of genistein in Escherichia coli.

Genistein, the main isoflavone in soy, has received considerable attention for its potential anti-carcinogenic properties. In a previous report, we investigated the possible role of genistein in anti-mutagenesis, using an Escherichia coli reversion assay system. Genistein reduced ENU-induced mutagenesis in a dose-dependent manner and the reduction of mutation frequency was differential among several categories of mutation. Most notable was a loss of transversion mutations, which require SOS functions. In this report, we further investigated the anti-mutagenic effect of genistein using a genetic approach. E. coli strains having alterations in genes involved in SOS-mutagenesis were examined, as were strains having defects in proteins that might serve as potential targets for genistein. The results showed that ENU-induced mutations produced in recA730 and lexA(Def) strains, both expressing a constitutive SOS response, were reduced by genistein to a lesser extent than in the wild-type strain. The effect of genistein was not entirely abolished, however. ENU mutagenesis in a umuC derivative, which reflects predominantly transition mutations, was unaffected by genistein. ENU-induced mutations in strains having defects in topA, gyrA, typA or uspA were not different than the wild-type, suggesting that these gene products were not involved in genistein's anti-mutagenic effect. In addition, we determined the distribution of genistein in various cellular fractions using HPLC. These studies revealed that genistein could be recovered from E. coli cells grown on agar media containing genistein; the intracellular concentration was similar to that in the agar plates. Further, most of the genistein recovered was associated with proteins in the cytosolic fraction and little partitioned in the membrane fraction. In vitro studies showed that genistein could be precipitated from a protein (BSA) containing solution. Finally, we examined the effect of genistein on formation of the RecA filament on ssDNA in vitro and observed an inhibition at high concentrations of genistein. In total, these results suggested that genistein may reduce SOS-dependent mutagenesis by reducing the interaction of RecA protein with ssDNA. As a consequence, genistein could cause a reduction in (1) the overall SOS response (confirmed using beta-galactosidase assays) and (2) trans-lesion DNA synthesis by DNA polymerase V.

Anticarcinogenic Agents↗
PubMed1998

A DNA self-assembled monolayer for the specific attachment of unmodified double- or single-stranded DNA.

A novel method for DNA surface immobilization and a paradigm for the attachment of unmodified DNA of any length or sequence are described herein. The development of a DNA self-assembled monolayer (DNA-SAM) that incorporates a DNA-thiol into a monolayer of inert alkane thiolates is reported. This DNA-SAM specifically hybridized complementary oligonucleotides while resisting the nonspecific adsorption of noncomplementary DNA and irrelevant proteins. Duplex DNA, having a single-stranded "capture tail," specifically bound to the DNA-SAM if the sequence of the "tail" was complementary to DNA presented in the SAM. The sense strand of the hybridized duplex DNA could be covalently attached to the surface by an enzymatic ligation reaction (leaving the anti-sense strand dissociable). DNA-binding proteins specifically bound to these surfaces only if their cognate sites were present in the duplex DNA.

Adsorption↗
PubMedDate not supplied

A common regulatory region shared by divergently transcribed genes of the Escherichia coli SOS system.

The Escherichia coli single-stranded DNA binding protein (SSB) is implicated in DNA replication, recombination and repair. On the chromosome, the ssb gene is located adjacent to the excision repair gene uvrA, but the two genes are transcribed in opposite directions. uvrA has been shown to be part of the E. coli SOS system by introducing Mud(Ap, lac) insertions distal to the regulatory region of the gene in the chromosome. Recent investigations suggest that SSB is also involved in the SOS response. However, because the SSB protein is essential to the cell, the inducibility of the ssb gene cannot be investigated by the insertion method. Therefore, we used plasmids harbouring the regulatory region of ssb fused to the galK structural gene, while leaving an intact ssb gene in the chromosome. We show here that expression of the ssb gene is dependent on two promoters of which one is damage inducible. Evidence is presented that the divergently transcribed ssb and uvrA genes are controlled by a common LexA binding site.

Bacterial Proteins↗
PubMed2000

The receptor tyrosine phosphatase-like protein ICA512 binds the PDZ domains of beta2-syntrophin and nNOS in pancreatic beta-cells.

Islet cell autoantigen (ICA) 512 of type I diabetes is a receptor tyrosine phosphatase-like protein associated with the secretory granules of neurons and endocrine cells including insulin-secreting beta-cells of the pancreas. Here we show that in a yeast two-hybrid assay its cytoplasmic domain binds beta2-syntrophin, a modular adapter which in muscle cells interacts with members of the dystrophin family including utrophin, as well as the signaling molecule neuronal nitric oxide synthase (nNOS). The cDNA isolated by two-hybrid screening corresponded to a novel beta2-syntrophin isoform with a predicted molecular mass of 28 kDa. This isoform included the PDZ domain, but not the C-terminal region, which in full-length beta2-syntrophin is responsible for binding dystrophin-related proteins. In vitro binding of the beta2-syntrophin PDZ domain to ICA512 required both ICA512's C-terminal region and an internal polypeptide preceding its tyrosine phosphatase-like domain. Immunomicroscopy and co-immunoprecipitations from insulinoma INS-1 cells confirmed the occurrence of ICA512-beta2-syntrophin complexes in vivo. ICA512 also interacted in vitro with the PDZ domain of nNOS and ICA512-nNOS complexes were co-immunoprecipitated from INS-1 cells. Finally, we show that INS-1 cells, like muscle cells, contain beta2-syntrophin-utrophin oligomers. Thus, we propose that ICA512, through beta2-syntrophin and nNOS, links secretory granules with the actin cytoskeleton and signaling pathways involving nitric oxide.

Alleles↗
PubMed1986

Mutagenic DNA repair in Escherichia coli. XII. Ultraviolet mutagenesis in excision-proficient umuC and lexA (ind-) bacteria as revealed by delayed photoreversal.

Streptomycin-resistant mutations are induced in excision-proficient umuC122::Tn5 bacteria given delayed photoreversal after u.v. light. The mutations occur after much earlier photoreversal than is found with the similar induction of tryptophan-independent mutations in excision-deficient umuC bacteria and the process is not markedly influenced by the presence of Casamino acids. The yield of mutants is between 25 and 40% of that found immediately after u.v. irradiation of isogenic umu+ bacteria. Mutagenesis is not inhibited by the presence of chloramphenicol after u.v. and before photoreversal when the bacteria have also been pretreated with chloramphenicol. Loss of photoreversibility of streptomycin-resistant mutations in umu+ excision-proficient bacteria also occurs during a similar period after u.v. and is similarly unaffected by Casamino acids and chloramphenicol. The results are interpreted on a 2-step model of error-prone repair in which a misincorporation step is followed by a lesion bypass step which requires induced levels of umuC gene product, the latter step being unnecessary when the pyrimidine dimer is removed by photoreversal after the misincorporation has taken place. Differences from the same phenomenon in excision-deficient bacteria are accounted for by the fact that in excision-proficient bacteria the mutation process is initiated by excision repair rather than during normal chromosomal replication. It is suggested that loss of photoreversibility in excision-proficient umu+ bacteria may reflect the misincorporation step only, thus explaining the apparent non-inducible nature of loss of photoreversibility previously reported. The bypass step alone requires induced levels of umuD,C gene products as is evidenced by the u.v.-induction of mutations in lexA102(ind-) bacteria after delayed photoreversal.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1995

Non-reciprocal regulation of Rhodobacter capsulatus and Rhodobacter sphaeroides recA genes expression.

The Rhodobacter capsulatus recA gene has been isolated and sequenced. Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity). However, the promoter regions of both R. capsulatus and R. sphaeroides recA genes are only 64% similar. An Escherichia coli-like LexA binding site was not present in the upstream region of the R. capsulatus recA gene. Nevertheless, the R. capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions. The R. capsulatus recA gene is poorly induced when inserted into the chromosome of R. sphaeroides, indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship.

Amino Acid Sequence↗
PubMed2003

Repair system for noncanonical purines in Escherichia coli.

Exposure of Escherichia coli strains deficient in molybdopterin biosynthesis (moa) to the purine base N-6-hydroxylaminopurine (HAP) is mutagenic and toxic. We show that moa mutants exposed to HAP also exhibit elevated mutagenesis, a hyperrecombination phenotype, and increased SOS induction. The E. coli rdgB gene encodes a protein homologous to a deoxyribonucleotide triphosphate pyrophosphatase from Methanococcus jannaschii that shows a preference for purine base analogs. moa rdgB mutants are extremely sensitive to killing by HAP and exhibit increased mutagenesis, recombination, and SOS induction upon HAP exposure. Disruption of the endonuclease V gene, nfi, rescues the HAP sensitivity displayed by moa and moa rdgB mutants and reduces the level of recombination and SOS induction, but it increases the level of mutagenesis. Our results suggest that endonuclease V incision of DNA containing HAP leads to increased recombination and SOS induction and even cell death. Double-strand break repair mutants display an increase in HAP sensitivity, which can be reversed by an nfi mutation. This suggests that cell killing may result from an increase in double-strand breaks generated when replication forks encounter endonuclease V-nicked DNA. We propose a pathway for the removal of HAP from purine pools, from deoxynucleotide triphosphate pools, and from DNA, and we suggest a general model for excluding purine base analogs from DNA. The system for HAP removal consists of a molybdoenzyme, thought to detoxify HAP, a deoxyribonucleotide triphosphate pyrophosphatase that removes noncanonical deoxyribonucleotide triphosphates from replication precursor pools, and an endonuclease that initiates the removal of HAP from DNA.

Adenine↗
PubMed2005

SOS-independent induction of dinB transcription by beta-lactam-mediated inhibition of cell wall synthesis in Escherichia coli.

Transcription of the dinB gene, encoding DNA polymerase IV, is induced by the inhibition of cell wall synthesis at different levels. Using the beta-lactam antibiotic ceftazidime, a PBP3 inhibitor, as a model, we have shown that this induction is independent of the LexA/RecA regulatory system. Induction of dinB transcription mediated by ceftazidime produces an increase in the reversion of a +1 Lac frameshift mutation.

Adenosine Triphosphatases↗
PubMed2002

Role for the adaptor protein Grb10 in the activation of Akt.

Grb10 is a member of the Grb7 family of adapter proteins lacking intrinsic enzymatic function and encodes functional domains including a pleckstrin homology (PH) domain and an SH2 domain. The role of different Grb10 splice variants in signal transduction of growth factors like insulin or insulin-like growth factor has been described as inhibitory or stimulatory depending on the presence of a functional PH and/or SH2 domain. Performing a yeast two-hybrid screen with the c-kit cytoplasmic tail fused to LexA as a bait and a mouse embryo cDNA library as prey, we found that the Grb10 SH2 domain interacted with the c-kit receptor tyrosine kinase. In the course of SCF-mediated activation of c-kit, Grb10 is recruited to the c-kit receptor in an SH2 domain- and phosphotyrosine-dependent but PH domain-independent manner. We found that Akt and Grb10 form a constitutive complex, suggesting a role for Grb10 in the translocation of Akt to the cell membrane. Indeed, coexpression studies revealed that Grb10 and c-kit activate Akt in a synergistic manner. This dose-dependent effect of Grb10 is wortmannin sensitive and was also seen at a lower level in cells in which c-kit was not expressed. Expression of a Grb10 mutant lacking the SH2 domain as well as a mutant lacking the PH domain did not influence Akt activity. Grb10-induced Akt activation was observed without increased phosphatidylinositol 3-kinase (PI3-kinase) activity, suggesting that Grb10 is a positive regulator of Akt downstream of PI3-kinase. Significantly, deficient activation of Akt by a constitutively activated c-kit mutant lacking the binding site for PI3-kinase (c-kitD814V/Y719F) could be fully compensated by overexpression of Grb10. In Ba/F3 cells, the incapacity of c-kitD814V/Y719F to induce interleukin-3 (IL-3)-independent growth could be rescued by overexpression of Grb10. In contrast, expression of the SH2 deletion mutant of Grb10 together with c-kitD814V/Y719F did not render Ba/F3 cells independent of IL-3. In summary, we provide evidence that Grb10 is part of the c-kit signaling pathway and that the expression level of Grb10 critically influences Akt activity. We propose a model in which Grb10 acts as a coactivator for Akt by virtue of its ability to form a complex with Akt and its SH2 domain-dependent translocation to the cell membrane.

Androstadienes↗
PubMed1993

Synergism between Tat and VP16 in trans-activation of HIV-1 LTR.

When tethered to heterologous DNA both Tat and VP16 can activate transcription from the HIV-1 LTR. To determine if they act by similar mechanisms, we constructed several hybrid effectors between Tat or VP16 and DNA-binding domains of GAL4 or LexA proteins. We tested these effectors on substituted reporter targets, which contained one to six GAL4 or LexA DNA-binding sites placed upstream of the HIV-1 promoter. Whereas Tat acted very inefficiently via DNA even with five DNA-binding sites, effects of VP16 were observed with a single DNA-binding site and increased with increasing number of sites. More importantly, effects of VP16 via DNA were synergistic with those of Tat via TAR RNA when both proteins were expressed simultaneously. We next created a tripartite fusion protein, which contained the GAL4 DNA-binding domain and activation domains of both Tat and VP16, which could be targeted to the HIV-1 LTR either via DNA or RNA. By introducing individual deleterious mutations into either Tat or VP16, we confirmed that effects of VP16 predominated via DNA whereas Tat but not VP16 acted via TAR RNA. Thus, Tat and VP16 act at different steps of the transcription process and increase expression from the HIV-1 LTR by different mechanisms.

Bacterial Proteins↗