Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli

Witkin, Evelyn M.; McCall, Jennifer; Volkert, Michael R.; Wermundsen, Ingbritt E.

doi:10.1007/bf00333788

Cited by 125 publications

(76 citation statements)

References 39 publications

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“…The RecA protein encoded by recA 718 causes moderate UV sensitivity but is competent in recombination, in SOS induction, and in all known RecAmediated proteolytic activities (10,17). We found that recA 718 polAI mutants were conditionally inviable.…”

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confidence: 74%

Overproduction of DnaE protein (alpha subunit of DNA polymerase III) restores viability in a conditionally inviable Escherichia coli strain deficient in DNA polymerase I

Witkin

Roegner-Maniscalco

1992

J Bacteriol

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Table 1 compares the growth of the double-mutant strain SC18-12 with that of each of the single-mutant strains and with that of a poLA12 strain from which recA has been deleted. ThepoLA12 recA 718 strain differed from each of the single mutants at 42°C in its inability to form colonies on nutrient agar and in its extremely slow growth on minimal medium. Similar results were obtained at 37°C. The polA12 recA-deleted strain was unconditionally inviable at 42°C and grew very poorly at 30°C. Figure 1 shows that the rate of DNA synthesis on nutrient agar, which even at 30°C was slightly slower in the recA718poLA12 strain SC18-12 than in either single mutant, decreased steadily in the double mutant after 1 h at 42°C. The rates of DNA synthesis in both single mutants were indistinguishable from those in the recA+ poU+ strain SC18-RP under the same conditions (data not shown).

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mentioning

confidence: 74%

Overproduction of DnaE protein (alpha subunit of DNA polymerase III) restores viability in a conditionally inviable Escherichia coli strain deficient in DNA polymerase I

Witkin

Roegner-Maniscalco

1992

J Bacteriol

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“…We also investigated the orientation bias for the SOS mutator effect, as mediated by the constitutive recA730 allele. In recA730 strains, the SOS system is induced constitutively, leading to increased mutagenesis in the absence of DNA-damaging treatments (untargeted mutagenesis) (10,39,40). We found, interestingly, that the orientation bias is inversed under these conditions and that, apparently, lagging strand replication is the major source of the mutations (22).…”

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confidence: 75%

“…This increased mutagenesis has been called the "SOS mutator activity" or "untargeted mutagenesis" (39,40). Like targeted mutagenesis, the SOS mutator activity depends on the umuDC genes (5).…”

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confidence: 99%

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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“…Bacterial Strains and Media-The strain used for isolating AZTresistant mutants is known as SC18-12 and has the genotype recA718 polA12 uvrA155 trpE65 lon-11 sul A1 (11). The strain BL21 DE3 was used for protein expression and has genotype FϪ ompT hsdSB(rBmBϪ) gal dcm (DE3).…”

Section: Methodsmentioning

confidence: 99%

3′-Azido-3′-deoxythymidine-resistant Mutants of DNA Polymerase β Identified by in Vivo Selection

Kosa

Sweasy

1999

Journal of Biological Chemistry

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We developed an in vivo selection to identify 3-azido-3-deoxythymidine (AZT)-resistant mutants of rat DNA polymerase ␤ (pol ␤). The selection utilizes pol ␤'s ability to substitute for Escherichia coli DNA polymerase I (pol I) in the SC18-12 strain, which lacks active pol I. pol ␤ allows SC18-12 cells to grow, but they depend on pol ␤ activity, so inhibition of pol ␤ by AZT kills them. We screened a library of randomly mutated pol ␤ cDNA for complementation of the pol I defect in the presence of AZT, and identified AZT-resistant mutants. We purified two enzymes with nonconservative mutations in the palm domain of the polymerase. The substitutions D246V and R253M result in reductions in the steadystate catalytic efficiency (K cat /K m ) of AZT-TP incorporation. The efficiency of dTTP incorporation was unchanged for the D246V enzyme, indicating that the substantial decrease in AZT-TP incorporation is responsible for its drug resistance. The R253M enzyme exhibits significantly higher K m (dTTP) and K cat (dTTP) values, implying that the incorporation reaction is altered. These are the first pol ␤ mutants demonstrated to exhibit AZT resistance in vitro. The locations of the Asp-246 and Arg-253 side chains indicate that substrate specificity is influenced by residues distant from the nucleotide-binding pocket.Efficient and accurate synthesis of DNA, during replication and repair, is essential to the integrity of any genome. Template-directed synthesis requires that a polymerase select the appropriate deoxynucleotide triphosphate (dNTP) and exclude incorrect bases. The interaction between a polymerase and substrates must therefore be highly specific, yet flexible, in order to maintain sequence fidelity. The smallest of the eukaryotic enzymes that accomplish this reaction is DNA polymerase ␤ (pol ␤), 1 a mammalian polymerase which fills short gaps in DNA (1). pol ␤ has been implicated in base excision repair (2, 3) and meiosis (4). pol ␤ is highly suitable for structure-function studies of the molecular mechanism of DNA synthesis due to the availability of information on the polymerase-DNA-substrate ternary complex (5, 6).DNA synthesis by pol ␤ can be compromised by the nucleoside analog drug AZT, which closely resembles a normal substrate. AZT-triphosphate (AZT-TP) presents a normal thymine moiety which may form a Watson-Crick base pair with adenosine, but has a modified sugar ring that results in chain termination. pol ␤ incorporates AZT into DNA in vitro (7) implying that pol ␤ is not able to distinguish perfectly between the drug and the natural nucleotide substrate. This susceptibility of pol ␤ makes AZT resistance a useful probe of enzyme-substrate interactions involved in DNA synthesis.The molecular basis for polymerase substrate specificity, and specifically AZT discrimination, is not well understood. The clinical problem of AZT-resistant HIV has been attributed to mutations in HIV reverse transcriptase. However, the mutant reverse transcriptase enzymes have exhibited little or no change in AZT-TP incorporation...

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Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli

Cited by 125 publications

References 39 publications

Overproduction of DnaE protein (alpha subunit of DNA polymerase III) restores viability in a conditionally inviable Escherichia coli strain deficient in DNA polymerase I

Overproduction of DnaE protein (alpha subunit of DNA polymerase III) restores viability in a conditionally inviable Escherichia coli strain deficient in DNA polymerase I

Lack of Strand Bias in UV-Induced Mutagenesis inEscherichia coli

3′-Azido-3′-deoxythymidine-resistant Mutants of DNA Polymerase β Identified by in Vivo Selection

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