Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The polB gene of Escherichia coli encodes DNA polymerase II whose role in vivo is not defined. The polB gene has been cloned and shown to be identical to a DNA damage-inducible gene dinA which is regulated by the LexA repressor. Nucleotide sequencing of polB reveals that E coli DNA polymerase II is highly homologous to replicative DNA polymerases of eukaryotes which include human DNA polymerase alpha and Saccharomyces cerevisiae DNA polymerases I, II and III. The polB gene is not required for growth, UV-repair and UV-mutagenesis.
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PMID:SOS-inducible DNA polymerase II of E coli is homologous to replicative DNA polymerase of eukaryotes. 191 43

The Escherichia coli polB gene encodes DNA polymerase II and is regulated by the SOS system. We sequenced a 4081 nucleotide segment of the E. coli chromosome that contains the polB gene and its flanking regions. DNA polymerase II, as deduced from the DNA sequence, consists of 782 amino acids, has a molecular weight of 89,917, and is structurally homologous to alpha-like DNA polymerases, which include eukaryotic replicative DNA polymerases. Comparison of the sequences of the alpha-like DNA polymerases including E. coli DNA polymerase II showed that there were nine highly conserved regions, and we constructed an unrooted phylogenetic tree of the DNA polymerases based on the differences in these conserved regions. The DNA polymerases of herpes groups viruses and the DNA polymerases that use protein priming for the initiation of replication form two separate subfamilies that occupy opposite locations in the tree. Other DNA polymerases, including E. coli DNA polymerase II, human DNA polymerase alpha, and yeast DNA polymerase I, occupy the central regions between the two subfamilies and they are rather distantly related to each other. The transcription initiation site of polB was identified by analysis of in vivo transcripts, and the promoter was assigned upstream of the polB coding region. The recognition sequence of the LexA repressor (SOS box) was identified by a footprinting experiment. It overlaps the -35 sequence of the polB promoter.
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PMID:Escherichia coli DNA polymerase II is homologous to alpha-like DNA polymerases. 203 16

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli produces a marked elevation in mutation rate. The mutation pathway is a component of the induced, multigene SOS response. SOS mutagenesis is a tightly regulated process dependent on two RecA-mediated proteolytic events: cleavage of the LexA repressor to induce the UmuC and UmuD mutagenesis proteins, and cleavage of UmuD to UmuD' to activate the mutation pathway. To investigate the protein-protein interactions responsible for SOS mutagenesis, we have studied the interaction of UmuC, UmuD, and UmuD'. To probe intracellular interaction, we have used immunoprecipitation techniques with antibodies against UmuC or UmuD and UmuD'. We have found that antibody to UmuC precipitates UmuD' from cell extracts, and antibody to UmuD and UmuD' precipitates UmuC. Thus we conclude that UmuC probably associates tightly with UmuD' in cells. For biochemical studies, we have purified the UmuC and UmuD' proteins to use with the previously purified UmuD. UmuC associates strongly with an affinity column of UmuD and UmuD', eluting only under strongly dissociating conditions (2 M urea or 1.5 M KSCN). UmuC also associates efficiently with UmuD or UmuD' in solution, as judged by velocity sedimentation in a glycerol gradient. The likely stoichiometry is one UmuC with a dimeric UmuD or UmuD'. From these experiments and previous work, we infer that SOS mutagenesis depends on the action of the UmuC-UmuD' complex and probably RecA to rescue a stalled DNA polymerase III holoenzyme at the DNA lesion.
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PMID:UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD'. 255 36

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.
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PMID:RecA protein and SOS. Correlation of mutagenesis phenotype with binding of mutant RecA proteins to duplex DNA and LexA cleavage. 296 Aug 17

We have investigated the ability of DNA polymerases from SOS-induced and uninduced Escherichia coli to incorporate nucleotides at a well-defined abasic (apurinic/apyrimidinic) DNA template site and to extend these chains from this unpaired 3' terminus. A DNA polymerase activity has been purified from E. coli, deleted for DNA polymerase I, that appears to be induced 7-fold in cells following treatment with nalidixic acid. Induction of this polymerase (designated DNA polymerase X) appears to be part of the SOS response of E. coli since it cannot be induced in strains containing a noncleavable form of the LexA repressor (Ind-). The enzyme is able to incorporate nucleotides efficiently opposite the abasic template lesion and to continue DNA synthesis. Although we observe an approximate 2-fold induction of DNA polymerase III in cells treated with nalidixic acid, several lines of evidence argue that DNA polymerase X is unrelated to DNA polymerase III (pol III). In contrast to pol X, pol III shows almost no detectable ability to incorporate at or extend beyond the abasic site; incorporation efficiency at the abasic lesion is at least 100-fold larger for pol X compared to pol III holoenzyme, pol III core, or pol III* (the polymerase III holoenzyme subassembly lacking the beta subunit). Pol X does not cross-react with polyclonal antibody directed against pol III holoenzyme complex or with monoclonal antibody prepared to the alpha subunit of pol III. Despite these structural and biochemical differences, pol X appears to interact specifically with the beta subunit of the pol III holoenzyme in the presence of single-stranded binding protein. Pol X has a molecular mass of 84 kDa. Our results indicate that this novel activity is likely to be identical to DNA polymerase II of E. coli.
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PMID:Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA. 305 91

The RecA protein of Escherichia coli is required for SOS-induced mutagenesis in addition to its recombinational and regulatory roles. Most SOS-induced mutations probably occur during replication across a DNA lesion (targeted mutagenesis). We have suggested previously that RecA might participate in targeted mutagenesis by binding preferentially to the site of the DNA damage (e.g., pyrimidine dimer) because of its partially unwound character; DNA polymerase III (polIII) will then encounter RecA-coated DNA at the lesion and might replicate across the damaged site with reduced fidelity. In this report, we analyze at a biochemical level two major predictions of this model. With respect to lesion recognition, we show that purified RecA protein binds more efficiently to UV-irradiated double-stranded DNA than to nonirradiated DNA, as judged by filter-binding and gel electrophoresis assays. With respect to replication fidelity, Fersht and Knill-Jones [Fersht, A. R. & Knill-Jones, J. W. (1983) J. Mol. Biol. 165, 669-682] have found that RecA inhibits the 3'----5' exonuclease (editing function) of polIII holoenzyme. We extend this observation by demonstrating that RecA inhibits the exonuclease of the purified editing subunit of polIII, epsilon protein. Thus, we suggest that the activities of RecA required for targeted mutagenesis are lesion-recognition, followed by localized inhibition of the editing capacity of the epsilon subunit of polIII holoenzme. In this proposed mechanism, one activation signal for RecA for mutagenesis is the lesion itself. Because UV-irradiated, double-stranded DNA efficiently activates RecA for cleavage of the LexA repressor, the lesion itself may also often serve as an activation signal for induction of SOS-controlled genes.
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PMID:Capacity of RecA protein to bind preferentially to UV lesions and inhibit the editing subunit (epsilon) of DNA polymerase III: a possible mechanism for SOS-induced targeted mutagenesis. 345 59

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst. From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication. A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells. In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced. We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.
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PMID:Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light. 623 Apr 59

Exposure of Escherichia coli to UV irradiation or nalidixic acid, which induce both the SOS and heat shock responses, led to a 3-4-fold increase in the amount of the beta subunit of DNA polymerase III holoenzyme, as assayed by Western blot analysis using anti-beta antibodies. Such an induction was observed also in a delta rpoH mutant lacking the heat shock-specific sigma 32 subunit of RNA polymerase, but it was not observed in recA13 or lexA3 mutants, in which the SOS response cannot be induced. Mapping of transcription initiation sites of the dnaN gene, encoding the beta subunit, using the S1 nuclease protection assay showed essentially no induction of transcription upon UV irradiation, indicating that induction is regulated primarily at the post-transcriptional level. Analysis of translational gene fusions of the dnaN gene, encoding the beta subunit, to the lacZ reporter gene showed induction of beta-galactosidase activity upon UV irradiation of cells harboring the fusion plasmids. Elimination of a 5' flanking DNA sequence in which the dnaN promoters P1 and P2 were located, did not affect the UV inducibility of the gene fusions. Thus, element(s) present from P3 downstream were sufficient for the UV induction. The induction of the dnaN-lacZ gene fusions was dependent on the recA and lexA gene products, but not on the rpoH gene product, in agreement with the immunoblot analysis. The dependence of dnaN induction on the SOS regulators was not mediated via classical repression by the LexA repressor, since the dnaN promoter does not contain a sequence homologous to the LexA binding site, and dnaN mRNA was not inducible by UV light. This suggests that SOS control may be imposed indirectly, by a post-transcriptional mechanism. The increased amount of the beta subunit is needed, most likely, for increased replication and repair activities in cells which have been exposed to UV radiation.
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PMID:Beta subunit of DNA polymerase III holoenzyme is induced upon ultraviolet irradiation or nalidixic acid treatment of Escherichia coli. 751 86

Rational thinking builds on feelings, too. This article starts with a tribute to Richard Setlow, an eminent scientist; it retraces as well some studies in molecular genetics that helped to understand basic questions of radiation biology. In the mid-1950s, the induction of a dormant virus (prophage) by irradiation of its host was an intriguing phenomenon. Soon, it was found that prophage induction results from the inactivation of the prophage repressor. Similarly, a score of induced cellular SOS functions were found to be induced when the LexA repressor is inactivated. Repressor inactivation involves the formation of a newly formed distinctive structure: a RecA-polymer wrapped around single-stranded DNA left by the arrest of replication at damaged sites. By touching this RecA nucleofilament, the LexA repressor is inactivated, triggering the sequential expression of SOS functions. The RecA nucleofilament acts as a chaperone, allowing recombinational repair to occur after nucleotide excision repair is over. The UmuD'C complex, synthesized slowly and parsimoniously, peaks at the end of recombinational repair, ready to be positioned at the tip of a RecA nucleofilament, placing the UmuD'C complex right at a lesion. At this location, UmuD'C prevents recombinational repair, and now acts as an error-prone paucimerase that fills the discontinuity opposite the damaged DNA. Finally, the elimination of lesions from the path of DNA polymerase, allows the resumption of DNA replication, and the SOS repair cycle switches to a normal cell cycle.
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PMID:At the birth of molecular radiation biology. 1174 47

The capacity of DNA macroarrays that contain all 4290 predicted open reading frames of the E. coli K12 genome was evaluated by measuring changes in gene expression in response to irradiation by ultraviolet light (UV). UV and other DNA damaging agents are known to trigger the induction of the SOS response. This is a coordinated increase in the level of expression of a set of approximately 30 unlinked genes, the SOS genes, negatively regulated by the LexA repressor. The analysis was performed on a set of isogenic strains with mutations that affect expression of genes of the SOS system: (i) the lexA+ strain, in which the SOS system can be induced after DNA damage, (ii) lexAind- mutants in which the SOS system cannot be induced, and (iii) lexAdef mutants in which the SOS system is induced constitutively. We found that a large set of genes appeared to be either upregulated or downregulated following UV irradiation. Among the genes which appeared to be upregulated in a LexA-dependent manner, we correctly identified 9 out of 27 SOS genes printed on the arrays and one gene containing a LexA binding site. One gene, dnaN, encoding the beta subunit of DNA polymerase III holoenzyme, was identified as an upregulated gene in a LexA-independent manner. Our results were compared to those of similar studies previously published. Although the SOS response as a whole could not be illustrated by using DNA arrays, the data suggest that regulation of some SOS genes might be more complex than previously thought.
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PMID:DNA array analysis of gene expression in response to UV irradiation in Escherichia coli. 1452 57


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