Different modes of loss of photoreversibility of mutation and lethal damage in ultraviolet-light resistant and sensitive bacteria

We used the lad forward mutagenesis system to determine the mutational specificity of UV-induced mutation in a repair-proficient (Uvr') and a repair-deficient (AUvrB) strain ofEscherichia coli. The spectra recovered at similar levels of mutagenesis were similar, the exception being a mutational hotspot at site A24 specific to the AUvrB strain. Mutations induced at this hotspot, as well as those induced at other mutational hotspots that were found to be common to both the Uvr' and Uvr-strains, involve G-C -* APT transitions. All of the hotspots are at sites of potential dipyrimidine photoproducts, such as thymine-cytosine and cytosine-cytosine dimers, or of pyrimidine-cytosine photoproduct Py-C* lesions. Each of these hotspots occurs at a site in the potential hairpin loop of quasi-palindromic sequences. These observations suggest an important role for DNA structure in determining the fate of UV-induced premutational lesions.The lacI system of E. coli provides a method for determining UV-induced mutational specificity at a large number of sites (1-3). In contrast, earlier studies in other systems generally relied upon the analysis ofreversion at a rather limited number of sites (4-6). Often the mutants analyzed in such reversion studies were originally induced by the mutagen (7); therefore, the possibility existed that preferentially mutable sites or hotspots had been selected and that these might behave atypically. Alternatively, the original mutation might have removed a critical DNA target sequence rendering the site practically immutable. Moreover, in studies of the reversion of nonsense mutations, the majority of "revertants" often occur not in the structural gene but at suppressor loci, which behave differently in their responses to UV light (4, 8, 9). The lacl system allows an examination of forward mutagenesis at 65 individual sites where nonsense mutations can arise by a single base substitution. Because both the DNA sequence and the location of the nonsense mutations have been established (10), each mutation can be attributed to a specific transition or transversion event.Detailed knowledge ofthe sites at which mutagenesis occurs and of the specific base changes produced may yield important clues about the premutagenic lesions and how they are processed. In the case ofUV irradiation, the mutational mechanism is poorly understood. Photoreactivation experiments suggest that mutagenesis in both excision-proficient and excision-deficient strains requires pyrimidine dimers (11,12). Nevertheless, considerable evidence exists that not all UV-induced mutagenesis occurs at the sites of induced lesions (13). For example, undamaged bacteriophage A plated on UV-irradiated hosts show high levels of recA+-dependent mutagenesis ("indirect mutagenesis") (14). In a current model, UV-induced mutagenesis in E. coli proceeds through a recA+-dependent, inducible error-prone repair pathway called "SOS" repair [reviewed by Witkin (13)]. It is postulated that, where alternative repair pathways are unable to function...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Mutational specificity of UV light in Escherichia coli: indications for a role of DNA secondary structure.

Todd¹,

Glickman²

1982

“…Thus, a progressive increase in the numbers of induced mutations that are not eliminated by photoreactivating light indicates the fixation of mutations into the cells' DNA by error-prone repair of pyrimidine dimer damage or the occurrence of an event which renders such fixation inevitable. Loss of photoreversibility of induced mutation to tryptophan prototrophy was used as a measure of the activity of error-prone repair and was determined by the method described by Nishioka and Doudney (29). Four-milliliter samples of irradiated culture were taken after various times of postirradiation induction with or without 50 Aig/ml of chloramphenicol.…”

Section: Methodsmentioning

confidence: 99%

Inducible error-prone repair in Escherichia coli.

Sedgwick¹

1975

104

A hypothesis that ultraviolet-induced mutagenesis arises from the induction of an error-prone mode of postreplication repair that reuires the exrA recA+ genotype has been tested with alkaline sucrose gradient centrifugation coupled with assays of fixation determined by loss of photoreversibility. The inhibitor of protein synthesis, chloramphenicol, added before irradiation, prevented a small amount of postreplication repair and completely eliminated mutation fixation in E. coil WP2S uvrA. However, chloramphenicol did not affect strand joining: (a) in uvrA bacteria allowed 20 min of growth between irradiation and antibiotic treatment; (b) in nonmutable uvrA exrA bacteria; and (c) in uvrA tif bacteria grown at 420 for 70 min before irradiation. These observations indicate that an inducible product is involved in a fraction of postreplication repair and is responsible for induced mutagenesis.A current hypothesis is that ultraviolet (UV)-induced mutagenesis in Escherichia coli is caused by an inducible, errorprone type of postreplication repair that requires the recA+ exrA + (or lexA+ ) genotype (1). Independently isolated exrA and lexA mutations have the same phenotype and map location and are thought to be mutations in the same cistron (2). In postreplication repair the DNA synthesized on a damaged template contains gaps (3). The frequency of gaps (4, 5) and indirect genetic (6-8) and biochemical (9) evidence indicate that gaps occur opposite lesions in the parental DNA. Gap closure in E. coli occurs through a recombinational exchange mechanism operating between daughter DNA duplexes (10, 11) and is slow enough so that rates of closure -may be measured by sedimentation techniques. Many exrA +recA+ -dependent phenomena, such as prophage induction (12-14), UV reactivation and mutagenesis of phage (15-17), and E. coil mutagenesis (16, 18, 19), exhibit common properties (1,15,(20)(21)(22), suggesting that they result from the induction of a single cellular process. Postreplication repair is also exrA + recA + -dependent (5,23,24) and might also require the same inducible functions. The error-prone nature of postreplication repair was proposed (18, 25) because of the increased mutability seen when the repair load carried by postreplication repair is increased by preventing excision repair (24,(26)(27)(28) and because reduced or no mutability is seen when the repair load is decreased by photoreactivation (24, 28). In addition, induced mutability (16,18,19) and postreplication repair of gaps are both prevented by exrA or recA mutations (5,23,24 (27), if induced mutagenesis requires induction of new gene products.These predictions were tested and confirmed in the experiments described below. MATERIALS AND METHODSStrains. E. coil B WP2S uvrA trp (26) and its conjugal and/or transductional derivatives WP67 uvrA polA trp (1) and CM611 uvrA exrA trp (30) have been described. WP44s-NF uvrA tif trp is a spontaneous nonfilamentous derivative of WP44s (22) which, unlike WP44s, retains viability at 420. I am extremely grat...

“…Genetic and nucleotide sequence analyses of mutated genes indicate that umuC' and umuD' gene products may act in a tolerance mechanism that permits synthesis of DNA on lesion-containing templates (18)(19)(20)(21)(22). Although the altered DNA synthesis might affect semiconservative replication, there is other evidence that it influences repair replication (23)(24)(25)(26). The sites of this activity might be a small fraction of gaps produced, either by excision repair or by chromosome replication as a prelude to postreplication repair.…”

mentioning

confidence: 99%

Differences in mutagenic and recombinational DNA repair in enterobacteria.

Sedgwick¹,

Goodwin²

1985

The incidence of recombinational DNA repair and inducible mutagenic DNA repair has been examined in Escherichia coli and 11 related species of enterobacteria. Recombinational repair was found to be a common feature of the DNA repair repertoire of at least 6 genera of enterobacteria. This conclusion is based on observations of (i) damageinduced synthesis of RecA-like proteins, (it) nucleotide hybridization between E. coli recA sequences and some chromosomal DNAs, and (iii) recA-negative complementation by plasmids showing SOS-inducible expression of truncated E. coli recA genes. The mechanism of DNA damage-induced gene expression is therefore sufficiently conserved to allow non-E. coli regulatory elements to govern expression of these cloned truncated E. coli recA genes. In contrast, the process of mutagenic repair, which uses umuC+ umuD+ gene products in E. coli, appeared less widespread. Little ultraviolet lightinduced mutagenesis to rifampicin resistance was detected outside the genus Escherichia, and even within the genus induced mutagenesis was detected in only 3 out of 6 species. Nucleotide hybridization showed that sequences like the E. coli umuCD+ gene are not found in these poorly mutable organisms. Evolutionary questions raised by the sporadic incidence of inducible mutagenic repair are discussed.The SOS system ofEscherichia coli is a sophisticated cellular response to DNA damage and involves induced synthesis of several DNA repair enzymes and changes in the normal cycles of cell division and replication. The key to the integration of these activities is a common transcriptional control mechanism in which expression of at least 17 genes is repressed by LexA A second role of RecA protein is in recombination. This activity is essential for both homologous recombination (6) and the major pathway of postreplication repair (7), which reconstitutes gapped daughter DNA strands by recombinational exchange (8,9). The large effect of this repair on survival is shown by the UV resistance of tsl-l and recA281 lexA mutants, which are repair proficient but do not induce expression of many SOS genes (10-13). Conversely, inhibition of recombination repair by recA-negative complementation causes radiosensitization without inhibiting induction of the SOS genes (14). recA-negative complementation can be caused by cloned truncated recA genes whose products are thought to impair the recombinational activity of chromosomally encoded recA+ protein by subunit mixing.The distinction between recombination repair and mutagenesis is best emphasized by the properties of umuCD mutants (15, 16). These mutants are deficient in mutagenesis induced by agents such as UV and are moderately radiosensitive. However, physical assays show that they are proficient in postreplication repair (17), which, as previously mentioned, is primarily recombinational. Genetic and nucleotide sequence analyses of mutated genes indicate that umuC' and umuD' gene products may act in a tolerance mechanism that permits synthesis of DNA on lesion-conta...