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PubMed1987

RecA protein and SOS. Correlation of mutagenesis phenotype with binding of mutant RecA proteins to duplex DNA and LexA cleavage.

The RecA protein of Escherichia coli is required for SOS-induced mutagenesis in addition to its recombinational and regulatory roles. We have suggested that RecA might participate directly in targeted mutagenesis by binding preferentially to the site of the DNA damage (e.g. pyrimidine dimer) because of its partially unwound nature; DNA polymerase III will then encounter RecA-coated DNA at the lesion and might replicate across the damaged site more often but with reduced fidelity. In support of this proposal, we have found that the phenotype of wild-type and mutant RecA for mutagenesis correlates with capacity to bind to double-stranded DNA. Wild-type RecA binds more efficiently to ultraviolet (u.v.)-irradiated, duplex DNA than to non-irradiated DNA. The RecA441 (Tif) protein that is constitutive for mutagenesis binds extremely well to double-stranded DNA with no lesions, whereas the RecA430 protein that is defective in mutagenesis binds poorly even to u.v.-irradiated DNA. The RecA phenotype also correlates with capacity to use duplex DNA as a cofactor for cleavage of the LexA repressor protein for SOS-controlled operons. Wild-type RecA provides efficient cleavage of LexA only with u.v.-irradiated duplex DNA; RecA441 cleaves well with non-irradiated DNA; RecA430 gives very poor cleavage even with u.v.-irradiated DNA. We conclude that the interaction of RecA with damaged double-stranded DNA is likely to be a critical component of SOS mutagenesis and to define a pathway for the LexA cleavage reaction as well.

Bacterial Proteins↗
PubMed1995

Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor.

The thyroid hormone (T3) receptors (TRs) are hormone-dependent transcription factors that regulate expression of a variety of specific target genes. To help elucidate the mechanisms that underlie this transcriptional regulation and other potential TR activities, we used the yeast interaction trap to isolate clones encoding proteins that specifically interact with the ligand binding domain of the rat TR beta. Several such proteins, called Trips (TR-interacting proteins), were isolated from independent selections carried out either in the presence or absence of T3. Surprisingly, all of the Trips were dependent on hormone for interaction with the TR, with some interacting only when T3 is present and others only when it is absent. Nearly all of the Trips also show similar ligand-dependent interaction with the retinoid X receptor (RXR), but none interact with the glucocorticoid receptor under any conditions. The sequences of three of the Trips predict specific functional roles: one is an apparent human homolog of a yeast transcriptional coactivator, one is a new member of a class of nonhistone chromosomal proteins, and one contains a conserved domain associated with ubiquitination of specific target proteins. Consistent with the pleiotropic effects of TR and RXR, several other Trips show significant amino acid sequence similarity with proteins involved in various regulatory pathways. The inherent transcriptional activity of the Trips was tested in yeast, and a chimeric protein consisting of a fusion of Trip4 to the bacterial LexA repressor protein is a relatively strong transcriptional activator. Similar LexA fusions to Trip9 and Trip10 had no transcriptional activity on their own but, when coexpressed with both TR and RXR, conferred T3-dependent activation to a reporter gene controlled by LexA binding sites. We suggest that this indirect T3 response provides a novel mechanism for hormonal activation of gene expression, and that studies of the Trips will provide important insights into the specific mechanisms of action of TRs and other receptors.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2004

LexA-DNA bond strength by single molecule force spectroscopy.

The SOS system of Escherichia coli is coordinated by two proteins: LexA, a repressor protein of several unlinked genes, and the coprotease RecA. As known to date LexA controls 31 genes with slightly different DNA binding motifs allowing for a variable degree of repression from one gene to the other. Besides the SOS system LexA plays an important role in the regulation of transcription. The protein regulates transcription by using particular motifs to bind DNA, the helix-turn-helix motif. Here, we employed AFM-based single molecule force spectroscopy to characterize the interaction of LexA protein with two different DNA motifs: recA and yebG. We measured the dissociation rates to be 0.045 s(-1) for recA and 0.13 s(-1) for yebG, respectively, which is in accordance with the predicted higher affinity between LexA-recA compared to LexA-yebG. The widths of the binding potentials were determined to be 5.4 +/- 1 angstroms and 4.9 +/- 0.5 angstroms, respectively. This short-ranged potential is characteristic for a stiff hydrogen-bonding network between protein and DNA. The unbinding occurs in a breakup rather than a gradual sliding.

Bacterial Proteins↗
PubMed1986

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.

Bacterial Proteins↗
PubMed1989

Interaction of the LexA repressor and the uvrC regulatory region.

We have studied the in vitro interaction of the LexA repressor protein and the uvrC regulatory region. We find that there is specific binding to two regions, the region we have defined as lexA1 and the lexA2-lexA3 region. Our findings support the possibility of an inducible regulation for this complex operon.

Bacterial Proteins↗
PubMed1988

RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis.

Induction of the Escherichia coli SOS system increases the ability of the cell to perform DNA repair and mutagenesis. Products of the recA and umuD,C genes are required for mutagenesis induced by radiation and many chemicals. Transcription of the SOS genes including recA and umuD,C is repressed by a repressor, LexA protein, and is derepressed by the proteolytic cleavage of LexA facilitated by RecA protein that had been activated by inducing signals produced in the cell by agents that damage DNA. An activated form of RecA protein, RecA, seems to have roles in SOS mutagenesis other than its known role as an antirepressor. Derepression of the genes involved in SOS mutagenesis such as recA and umuD,C in defective chromosomal lexA(Def) mutants does not increase the ability of the cell to perform mutagenesis. Activation of RecA protein is essential to this ability. RecA facilitates the proteolytic cleavage of several repressors such as lambda, P22, and 434 phage repressors and LexA, and UmuD protein contains a sequence homologous to the regions surrounding the cleavage sites of these repressors; therefore, we examined the possibility that UmuD protein is cleaved by RecA. We found evidence that the intact UmuD protein was cleaved after mutagenic treatment and that the cleavage was dependent on RecA. The results suggested that UmuD protein may be proteolytically processed by RecA, and that processed UmuD may be the active form of the protein participating in mutagenesis.

Bacterial Proteins↗
PubMed2002

Escherichia coli RecX inhibits RecA recombinase and coprotease activities in vitro and in vivo.

In Escherichia coli the RecA protein plays a pivotal role in homologous recombination, DNA repair, and SOS repair and mutagenesis. A gene designated recX (or oraA) is present directly downstream of recA in E. coli; however, the function of RecX is unknown. In this work we demonstrated interaction of RecX and RecA in a yeast two-hybrid assay. In vitro, substoichiometric amounts of RecX strongly inhibited both RecA-mediated DNA strand exchange and RecA ATPase activity. In vivo, we showed that recX is under control of the LexA repressor and is up-regulated in response to DNA damage. A loss-of-function mutation in recX resulted in decreased resistance to UV irradiation; however, overexpression of RecX in trans resulted in a greater decrease in UV resistance. Overexpression of RecX inhibited induction of two din (damage-inducible) genes and cleavage of the UmuD and LexA repressor proteins; however, recX inactivation had no effect on any of these processes. Cells overexpressing RecX showed decreased levels of P1 transduction, whereas recX mutation had no effect on P1 transduction frequency. Our combined in vitro and in vivo data indicate that RecX can inhibit both RecA recombinase and coprotease activities.

Adenosine Triphosphate↗
PubMed2005

Error-prone repair and translesion synthesis III: the activation of UmuD (or less is more).

Following DNA damage to Escherichia coli bacteria, RecA protein is activated by binding to single stranded DNA and cleaves its own gene repressor (LexA protein). Two papers from Graham Walker's laboratory showed that several bacterial genes in addition to RecA are repressed by the LexA repressor and are inducible following DNA damage [C.J. Keyon, G.C. Walker, DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli, in: Proceedings of the National Academy of Sciences of the United States of America 77, 1980, pp. 2819--2823] and predicted that one of them (UmuD) might itself be subject to activation by a further cleavage reaction involving activated RecA protein [K.L. Perry, S.J. Elledge, B.B. Mitchell, L. Marsh, G.C. Walker, umuD,C and mucA,B operans whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology, in: Proceedings of the National Academy of Sciences of the United States of America 82, 1985, pp. 4331--4335]. The processed form of UmuD, termed UmuD', later proved to be a subunit of DNA polymerase V, a key enzyme involved in translesion synthesis.

Bacterial Proteins↗
PubMed2000

Efficient repair of hydrogen peroxide-induced DNA damage by Escherichia coli requires SOS induction of RecA and RuvA proteins.

The survival of Escherichia coli following treatment with a low dose (1-3 mM) of hydrogen peroxide (H(2)O(2)) that causes extensive mode-one killing of DNA repair mutants is stimulated by the induction of the SOS regulon. Results for various mutants indicate that induction of recA and RecA protein-mediated recombination are critical factors contributing to the repair of H(2)O(2)-induced oxidative DNA damage. However, because DNA damage activates RecA protein's coprotease activity essential to cleavage of LexA repressor protein and derepression of all SOS genes, it is unclear to what extent induction of RecA protein stimulates this repair. To make this determination, we examined mode-one killing of DeltarecA cells carrying plasmid-borne recA (P(tac)-recA(+)) and constitutively expressing a fully induced level of wild-type RecA protein when SOS genes other than recA are non-inducible in a lexA3 (Ind(-)) genetic background or inducible in a lexA(+) background. At a H(2)O(2) dose resulting in maximal killing, DeltarecA lexA3 (Ind(-)) cells with P(tac)-recA(+) show 40-fold greater survival than lexA3 (Ind(-)) cells with chromosomal recA having a low, non-induced level of RecA protein. However, they still show 10- to 15-fold lower survival than wild-type cells and DeltarecA lexA(+) cells with P(tac)-recA(+). To determine if the inducible RuvA protein stimulates survival, we examined a ruvA60 mutant that is defective for the repair of UV-induced DNA damage. This mutant also shows 10- to 15-fold lower survival than wild-type cells. We conclude that while induction of RecA protein has a pronounced stimulatory effect on the recombinational repair of H(2)O(2)-induced oxidative DNA damage, the induction of other SOS proteins such as RuvA is essential for wild-type repair.

Bacterial Proteins↗
PubMed1993

Genes for the establishment and maintenance of lysogeny by the temperate coliphage 186.

To identify the genes in coliphage 186 that are required for lysogeny, we isolated clear-plaque mutants. Complementation studies and DNA sequencing identified two genes, the cI gene for the immunity maintenance repressor and the cII gene, which is required only for the establishment of lysogeny. One mutant carried a change in the LexA-binding site controlling expression of the antirepression protein Tum.

Bacterial Proteins↗
PubMed1992

Spontaneous transposition in the bacteriophage lambda cro gene residing on a plasmid.

A new mutagenesis assay system based on the phage lambda cro repressor gene residing on a plasmid was developed. The assay detects mutations in cro that decrease the binding of the repressor to the OR operator in an OR PR-lacZ fusion present in a lambda prophage. Mutations arose spontaneously during growth of E. coli cells harboring cro plasmids at a frequency of 3-6 x 10(-6). Analysis of some 200 cro mutants from several 'wild-type' strains revealed a substantial fraction of 25-70% insertion events caused by transposition of IS elements. Most of the insertions were caused by IS1, but IS5 insertions were observed too. In strains harboring Tn10, IS10 was responsible for most insertions. Restriction nuclease digestion analysis revealed a preference for insertion of IS10 into the C-terminal half of cro, despite the absence of sequences which are known hot spots for Tn10 insertions. The frequency of IS1 insertions into cro decreased 25-60-fold and that of IS10 insertions decreased 200-fold in cells carrying the recA56 mutation, suggesting that RecA is involved in transposition of these elements. During the logarithmic phase of growth, the mutation frequency was constant for at least 22 generations; however, upon continuous incubation at the stationary phase, the mutation frequency gradually increased, yielding a 3-fold increase in the frequency of insertion and a 4-5-fold increase in point mutation. Genomic Southern analysis of chromosomal IS elements in cells which underwent a transposition from the chromosome into the cro plasmid revealed that the number and distribution of IS1 and IS5 were usually unaltered compared to cells which did not undergo a transposition event. In contrast, essentially each IS10 transposition was accompanied by multiple events which led to changes in the number and distribution of chromosomal IS10 elements.

Adenosine Triphosphatases↗
PubMed1993

The LexA repressor binds within the deep helical groove of the activated RecA filament.

The RecA protein of Escherichia coli, as a result of DNA damage, catalyzes the cleavage of its own repressor, the LexA protein, and thereby initiates the SOS response. Using a non-cleavable LexA mutant, we have obtained a co-complex of both the RecA and LexA proteins on DNA. Mass analysis using scanning transmission electron microscopy suggests that the site size of the LexA repressor on RecA is two, which would be consistent with a nearest-neighbor exclusion model for binding. Three-dimensional reconstruction of electron micrographs of these filaments shows that the LexA protein is bound in the deep groove of the RecA filament, with two strong regions of contact that span adjacent RecA protomers within the filament. One contact is consistent with a proposed LexA binding site in the RecA crystal structure. The other contact maps onto a region that has been postulated to be a second DNA-binding site within RecA, which can explain the inhibition of RecA cleavage of LexA by excess DNA.

Bacterial Proteins↗
PubMed1989

Amino acid similarities to other proteins offer insights into roles of UmuD and UmuC in mutagenesis.

The products of the umuD and umuC genes are required for most uv and chemical mutagenesis in Escherichia coli. The genes are organized in an operon that is repressed by LexA and regulated as part of the SOS response. The umuD protein shares homology with the carboxyl-terminal domain of LexA. Genetic evidence now indicates that RecA-mediated cleavage activates UmuD for its role in mutagenesis. The COOH-terminal fragment of UmuD is both necessary and sufficient for this role. Similarities of UmuD to gene 45 protein of bacteriophage T4 and of UmuC to gene 44 protein and gene 62 protein suggest possible roles for UmuD and UmuC in mutagenesis that are supported by preliminary evidence.

Amino Acid Sequence↗
PubMed1983

Autoinduced synthesis of colicin E2.

Escherichia coli K-12 cells carrying the high copy number plasmid ColE2-P9 and a sfiA-lacZ gene fusion exhibit abnormally high levels of SOS-regulated phi sfiA-lacZ expression. Increased sfiA-lacZ expression is caused by the action of colicin E2, which is a DNase, rather than by the presence of multiple copies of a binding site for LexA protein, the repressor for the sfiA and colicin E2 genes. Expression of sfiA-lacZ was reduced to normal levels if the ColE2+ strain lacked the outer membrane colicin E2 receptor protein (BtuB) or if they carried an increased number of colicin E2 immunity genes. The results suggest that cultures of ColE2+ strains contain a small number of cells which produce colicin which can then enter other, non-producing cells in the culture and cause sufficient damage to the DNA to induce the SOS system. The levels of colicin E2 immunity in the producing cells is presumably sufficient to prevent extensive lethal effects of the colicin, but insufficient to prevent limited endonuclease activity. An important consequence of this phenomenon is that the DNase action of colicin E2 can stimulate its own production.

Bacterial Proteins↗
PubMed1985

Dual role for Escherichia coli RecA protein in SOS mutagenesis.

Induction of the Escherichia coli SOS system increases the ability of the cells to perform DNA repair and mutagenesis. Previous work has shown that this increased mutagenesis is the result of derepression of specific genes through a complex regulatory mechanism controlled by LexA and RecA proteins. One role of RecA protein in this process is to facilitate proteolytic cleavage of LexA protein (the repressor) in response to an inducing signal that reversibly activates RecA protein to perform this function. We show that activated RecA protein plays a second role in SOS mutagenesis, as revealed by analyzing repair of UV-damaged phage lambda in host mutants with alterations in the SOS regulatory system. First, phage mutagenesis was not expressed constitutively in a mutant that is derepressed through lack of functional LexA protein; activated RecA protein was still required. Second, phage mutagenesis was constitutively expressed in the presence of recA mutations that alter RecA protein so that it is activated in normally growing cells. There was also RecA-dependent constitutive expression of SOS mutagenesis in host mutants that lack functional LexA protein and carry plasmids. We discuss several possible biochemical mechanisms for this second role of activated RecA protein in SOS mutagenesis.

Bacterial Proteins↗
PubMed2000

Saccharomyces cerevisiae Ras/cAMP pathway controls post-diauxic shift element-dependent transcription through the zinc finger protein Gis1.

The Saccharomyces cerevisiae protein kinase Rim15 was identified previously as a component of the Ras/cAMP pathway acting immediately downstream of cAMP-dependent protein kinase (cAPK) to control a broad range of adaptations in response to nutrient limitation. Here, we show that the zinc finger protein Gis1 acts as a dosage-dependent suppressor of the rim15Delta defect in nutrient limitation-induced transcriptional derepression of SSA3. Loss of Gis1 results in a defect in transcriptional derepression upon nutrient limitation of various genes that are negatively regulated by the Ras/cAMP pathway (e.g. SSA3, HSP12 and HSP26). Tests of epistasis as well as transcriptional analyses of Gis1-dependent expression indicate that Gis1 acts in this pathway downstream of Rim15 to mediate transcription from the previously identified post-diauxic shift (PDS) element. Accordingly, deletion of GIS1 partially suppresses, and overexpression of GIS1 exacerbates the growth defect of mutant cells that are compromised for cAPK activity. Moreover, PDS element-driven expression, which is negatively regulated by the Ras/cAMP pathway and which is induced upon nutrient limitation, is almost entirely dependent on the presence of Gis1.

Bacterial Proteins↗
PubMed1982

Read-through transcription from a derepressed Tn3 promoter affects ColE1 functions on a ColE1::Tn3 composite plasmid.

Mutations in the repressor encoded by the transposon Tn3 tnpR gene lead to increased levels of expression of two gene products: the mutant repressor (TnpR-) and the Tn3 encoded transposase, TnpA (Heffron et al. 1978; Chou et al. 1979a). Derivatives of the ColE1::Tn3 composite plasmid, RSF2124, with mutant Tn3 repressor exhibited the expected elevated levels of transposition. Unexpectedly, hosts containing these tnpR- derivatives produced enhanced levels of the ColE1 encoded toxin, colicin E1. The gene for colicin E1 maps far (0.23-0.98 MU) from the Tn3 insertion point (0.73 MU) (Fig. 1). The colicin E1 overproduction phenotype, designated Eop-, was complemented in trans by wild type repressor gene product (TnpR+) to the wild type phenotype, Eop+. Hosts with RSF2124 derivatives which expressed high levels of both mutant repressor and mutant transposase (TnpR-, TnpA-) were Eop-. Hosts containing plasmids deleted for both tnpA and tnpR promoters were Eop+, while hosts with plasmids carrying a lac promoter substitution for the tnpA promoter were Eop-. These data support the idea that a cis-acting effect of increased transcription from the tnpA promoter into adjacent ColE1 DNA was the cause of colicin overproduction. Increased transcription activated a putative colicin augmentation function (caf) whose presence was required for the Eop- phenotype. Deletion mapping established that one boundary of the caf locus lies within 52 bases of the junction of the left end of Tn3 and ColE1 DNA. ColE1 DNA in this area contains an open reading frame which could encode either a 74 or a 63 residue protein (B. Polisky, unpublished DNA sequence data). The presence of increased levels of an mRNA transcript from this region and/or the increased expression of protein(s) from this transcript could result in an Eop- phenotype. Expression of the Eop- phenotype requires the presence of the host recE gene. Evidence is presented which suggests that the recA repressor, lexA protein, controls expression of the recE gene product, ExoVIII.

Cloning, Molecular↗