The “tale” of UmuD and its role in SOS mutagenesis†

Gonzalez, Martín; Woodgate, Roger

doi:10.1002/bies.10040

Cited by 28 publications

(23 citation statements)

References 87 publications

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“…Our data suggest that the access of Pol V to the primer terminus (the presumed rate-limiting step for translesion synthesis) is ultimately the same for either strand, despite the enzymological differences between the two replication modes. This may be consistent with the reported timing of UV mutagenesis, occurring some 45 to 60 min after the initial blockage of replication by the UV irradiation, as deduced from delayed photoreactivation experiments (3) and time course of UmuDC induction/ activation (16,31). At these later times, memory of leading versus lagging strand replication is not likely to be retained.…”

Section: Vol 184 2002 Uv Mutagenesis In E Coli 4451supporting

confidence: 87%

Lack of Strand Bias in UV-Induced Mutagenesis inEscherichia coli

et al. 2002

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We have investigated whether UV-induced mutations are created with equal efficiency on the leading and lagging strands of DNA replication. We employed an assay system that permits measurement of mutagenesis in the lacZ gene in pairs of near-identical strains. Within each pair, the strains differ only in the orientation of the lacZ gene with respect to the origin of DNA replication. Depending on this orientation, any lacZ target sequence will be replicated in one orientation as a leading strand and as a lagging strand in the other orientation. In contrast to previous results obtained for mutations resulting from spontaneous replication errors or mutations resulting from the spontaneous SOS mutator effect, measurements of UV-induced mutagenesis in uvrA strains fail to show significant differences between the two target orientations. These data suggest that SOS-mediated mutagenic translesion synthesis on the Escherichia coli chromosome may occur with equal or similar probability on leading and lagging strands.Exposure of cells to UV light results in the formation of mutagenic DNA lesions, among which the two most frequent lesions are cyclobutane pyrimidine dimers and 6-4 pyrimidinepyrimidone photoproducts at adjacent pyrimidines (13). In the bacterium Escherichia coli, mutagenesis by UV light and by a variety of chemical mutagens is mediated by the inducible SOS response (13,29). The SOS response is a global response involving the coordinated expression of some 30 genes (9). Its general function is to promote cellular survival upon induction of DNA damage, albeit at the cost of increased mutagenesis. The response is controlled by the interplay of the RecA and LexA proteins, the latter constituting the transcriptional repressor of the SOS regulon (13). After blockage of ongoing DNA replication by DNA damage, RecA forms nucleoprotein filaments with the damage-induced single-stranded DNA. This activated form of RecA then mediates the cleavage of LexA, thereby enabling the expression of the SOS genes.SOS mutagenesis is dependent on the lexA-controlled umuDC operon (17,18,32), which upon induction of the system yields the UmuDЈ 2 C complex (UmuDЈ being the RecAmediated cleavage product of UmuD). This complex was recently shown to be a DNA polymerase, termed Pol V (26,28,(34)(35)(36). Pol V is thought to promote mutagenesis by mediating mutagenic bypass of the DNA lesions, such as pyrimidine dimers, after DNA Pol III holoenzyme (HE) is blocked at such lesions. Pol V is a member of a recently described class of DNA polymerases (the UmuC/DinB/Rad30/Rev1 superfamily), now named Y-family DNA polymerases, that is found in a broad range of organisms (25). These enzymes are best characterized by their generally low-fidelity synthesis and/or ability to bypass DNA lesions in vitro (for a review, see reference 38). In addition to Pol V, E. coli also possesses another SOSinducible Y-family DNA polymerase, Pol IV (37). The precise functions of Pol IV are not yet clear, but its role in DNAdamage induced mutagenesis, including UV muta...

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Section: Vol 184 2002 Uv Mutagenesis In E Coli 4451supporting

confidence: 87%

Lack of Strand Bias in UV-Induced Mutagenesis inEscherichia coli

et al. 2002

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“…How common is cooperative stability in cells, and how readily can cooperative stability be implemented (e.g., evolved) molecularly if the need arises? We already mentioned in the introduction a number of specific examples (1)(2)(3)(4)(5)(6) where oligomerization provides protection against degradation. In these instances, the mechanisms of cooperative stability fell within the following two classes: (i) oligomerization enhances the thermal stability of the protein subunits, making them more resistant to proteolysis, and (ii) oligomerization buries the degradation tags that would have been exposed in monomeric subunits.…”

Section: Discussionmentioning

confidence: 99%

“…This effect, referred to below as ''cooperative stability,'' has been discussed previously in qualitative terms in the context of many well-studied examples in prokaryotes and eukaryotes (1,2). For example, in the SOS response of Escherichia coli, UmuC degradation is rescued by oligomerization with UmuDЈ 2 (3). Additionally, in Saccharomyces cerevisiae, the dimerization of a1 and ␣2 reduced the degradation rate by as much as 15-fold (4).…”

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confidence: 90%

Nonlinear protein degradation and the function of genetic circuits

Buchler

Gerland

Hwa

2005

Proc. Natl. Acad. Sci. U.S.A.

159

170

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The functions of most genetic circuits require a sufficient degree of cooperativity in the circuit components. Although mechanisms of cooperativity have been studied most extensively in the context of transcriptional initiation control, cooperativity from other processes involved in the operation of the circuits can also play important roles. In this work, we examine a simple kinetic source of cooperativity stemming from the nonlinear degradation of multimeric proteins. Ample experimental evidence suggests that protein subunits can degrade less rapidly when associated in multimeric complexes, an effect we refer to as ''cooperative stability.'' For dimeric transcription factors, this effect leads to a concentration-dependence in the degradation rate because monomers, which are predominant at low concentrations, will be more rapidly degraded. Thus, cooperative stability can effectively widen the accessible range of protein levels in vivo. Through theoretical analysis of two exemplary genetic circuits in bacteria, we show that such an increased range is important for the robust operation of genetic circuits as well as their evolvability. Our calculations demonstrate that a few-fold difference between the degradation rate of monomers and dimers can already enhance the function of these circuits substantially. We discuss molecular mechanisms of cooperative stability and their occurrence in natural or engineered systems. Our results suggest that cooperative stability needs to be considered explicitly and characterized quantitatively in any systematic experimental or theoretical study of gene circuits.amplification ͉ dimerization ͉ bistability ͉ oscillation I t is widely recognized that controlled proteolysis, where the degradation of one protein depends on the presence of another protein in the cell, can play an important regulatory role in genetic circuits (1). Here, we examine another effect of proteolysis that does not involve such regulatory control, but can nevertheless impact the function of genetic circuits in important ways. It is a kinetic, cooperative effect predicated on the following two essential ingredients: (i) the fact that many proteins perform their physiological functions as dimers or higher-order oligomers, and (ii) the tendency for the oligomers to be more stable (to proteolysis) than their monomeric components. This effect, referred to below as ''cooperative stability,'' has been discussed previously in qualitative terms in the context of many well-studied examples in prokaryotes and eukaryotes (1, 2). For example, in the SOS response of Escherichia coli, UmuC degradation is rescued by oligomerization with UmuDЈ 2 (3). Additionally, in Saccharomyces cerevisiae, the dimerization of a1 and ␣2 reduced the degradation rate by as much as 15-fold (4). Possible molecular mechanisms that give rise to cooperative stability include enhanced thermal stability of proteins upon mutual association [because thermal instability correlates with the rate of degradation (5, 6)] and the burial of proteolytic recogni...

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“…Characterized UmuD proteins are subunits of an error-prone DNA polymerase that participates in the repair of DNA damage during the SOS response (97). The E. coli UmuD protein is synthesized in an inactive form, and RecA-mediated autocleavage of its peptide backbone activates it to become a subunit of the UmuC/UmuDЈ error-prone DNA polymerase (39).…”

Section: Discussionmentioning

confidence: 99%

The pKO2 Linear Plasmid Prophage ofKlebsiella oxytoca

et al. 2004

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Temperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages N15 and PY54 are known to have a linear plasmid prophage with closed hairpin telomeres. We report here the complete nucleotide sequence of the 51,601-bp Klebsiella oxytoca linear plasmid pKO2, and we demonstrate experimentally that it is also a prophage. We call this bacteriophage KO2. An analysis of the 64 predicted KO2 genes indicate that it is a fairly close relative of phage N15; they share a mosaic relationship that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly identical DNA binding specificities. The KO2 virion is unusual in that its phage -like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The KO2 genome also carries putative homologues of bacterial dinI and umuD genes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated by LexA protein binding to a single target site that overlaps both promoters.Temperate bacteriophages usually physically integrate their prophage DNAs into the host bacterium's chromosome when they establish lysogeny. However, a few prophages, such as those of phages P1, N15, LE1, 20, and BB-1, are not integrated but replicate in the lysogen as low-copy-number plasmids (32,38,52,53,84). Of these, the Escherichia coli doublestranded DNA (dsDNA) tailed-phage N15 has an unusual linear prophage plasmid that has covalently closed hairpin telomeres (67, 68). Although N15 has been studied in some detail (83,85,87), few other phages with similar linear DNA prophages have been described. While this paper was being prepared, another linear plasmid prophage, PY54, was reported for Yersinia enterocolitica (48). Furthermore, the linear, hairpin-ended plasmids Lp28-2 and Lp54 in the spirochete Borrelia burgdorferi B31-MI carry sequence similarities to dsDNA phage virion assembly genes (19, 33); however, they have not yet been shown to be bone fide prophages. Linear plasmids are very rare in the ␥-Proteobacteria, but it has been reported that Klebsiella oxytoca strain CCUG 15788 (a nickelresistant bacterium isolated from the mineral oil-water coolant-lubricant emulsion of a metal working machine in Göte-borg, Sweden, in 1989 [69]) harbors a linear plasmid, pKO2, of about 50 kbp (95). Since this is close to the size of the N15 linear plasmid prophage, we were prompted to investigate the possibility that this plasmid could be a prophage. We report here the complete nucleotide sequence of pKO2, a linear plasmid prophage that encodes the synthesis of a tailed-phage particle, and the analysis of some of its properties. MATERIALS AND METHODSBacterial strains and plasmid construction. The bacterial st...

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The “tale” of UmuD and its role in SOS mutagenesis†

Cited by 28 publications

References 87 publications

Lack of Strand Bias in UV-Induced Mutagenesis inEscherichia coli

Lack of Strand Bias in UV-Induced Mutagenesis inEscherichia coli

Nonlinear protein degradation and the function of genetic circuits

The pKO2 Linear Plasmid Prophage ofKlebsiella oxytoca

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