Different modes of loss of photoreversibility of ultraviolet light-induced true and suppressor mutations to tryptophan independence in an auxotrophic strain of Escherichia coli

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%

“…Evidence has accumulated suggesting that pathways for UV-induced mutagenesis differ in excision-proficient and excision-deficient strains (11,17,18). In the case of repair-proficient strains, it is hypothesized that mutations arise at sites within structural genes during excision-repair gap filling.…”

mentioning

confidence: 99%

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

Todd¹,

Glickman²

1982

“…Many of the mutations that occur in wild-type cells become irreversible during the first 20 min after UV irradiation (30,31). Since the inducible, error-prone, SOS system is required for UV mutagenesis (32,33), a short delay in repair may result in substantially more photoproducts being present as SOS is being induced.…”

Section: Discussionmentioning

confidence: 99%

Excision repair at individual bases of the Escherichia coli lacI gene: relation to mutation hot spots and transcription coupling activity.

Subrahmanyam

Brash

1992

To determine whether base-to-base variations in the rate of excision repair influence the distribution of mutations, we have developed a method to measure UV photoproducts at individual nucleotides in the Escherichia coli chromosome. Specific gene fragments are 3' end-labeled using a sequence-specific oligonucleotide to direct the site of labeling, and photoproducts are identified by enzymatic incision. On the nontranscribed strand of the E. coli lWd gene, the cyclobutane pyrimidine dimer frequency was 2-to 8-fold higher in chromosomal DNA than in a cloned DNA fragment. The chromosomal lesion frequency corresponded to the frequency of UVinduced mutations at mutation hot spots reported in the literature. Only 0-30% of cyclobutane dimers at various sites on this strand were excised in 20 min. In contrast, repair on the transcribed strand was 80-90% complete in 20 min. However, the transcribed strand contained an excision repair "slow spot" at the site of its single mutation hot spot: At this site, no repair occurred for the first 10 min, after which repair proceeded more slowly than typical of that strand. In an mid strain, deficient in a factor that couples repair to transcription in cell extracts, the excision rate at individual nucleotides on the transcribed strand was minimal at most sites for at least 30 min.

“…MFD-susceptible suppressor mutations comprise the only class of, UV-induced mutations known to require semiconservative DNA replication for their fixation as stable genetic changes, >90%o of them remaining susceptible to elimination either by MFD-inducing posttreatments (29) or by photoreactivating light (30) until DNA replicates, indicating that they are caused by unexcised photoproducts. Other types of induced mutations in wildtype strains (true back-mutations to prototrophy, mutations to streptomycin resistance), which are an order of magnitude less frequent, in any medium, than suppressor mutations in rich medium, lose their photoreversibility much more rapidly (30)(31)(32). These mutations are believed to originate before replication, in genes from which all premutational photoproducts are excised before replication resumes, by error-prone SOS processing of a small number of excision gaps.…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli mfd mutant deficient in "mutation frequency decline" lacks strand-specific repair: in vitro complementation with purified coupling factor.

Selby

Witkin

Sancar

1991

199

109

Mutation frequency decline (NWD) is the rapid decrease in the frequency of certain induced nonsense suppressor mutations occtnng when protein sythesis translendy inhibited Immediately fter irrtin. MD is abolihed by mutations inthe uvrA, -B. or -C genes, which prevent excision repair, or by a mfd mutation, which reduces the rate ofexcision but does not affect survival. Using an in vitro repair synthesis assay we found that although wild-type ells repair the ibed (template) strand frentially, mfd cells are incapable of d-pcfic repar. The iciency in strand-selective repair of ,*d-cell extract was cor d by adding highiy purified "transcription-repair coupling fator" to the reaction mixt. We conclude that mfd is, most likely, the gene encoding the tnscription-repai coupling factor.In recent years in vivo studies have shown that, in general, actively transcribing genes are repaired at a faster rate than the rest of the genome (1-3). In the majority of the cases gene-specific 'repair appears to be due to strand-specific repair-that is, in an. actively transcribing gene the template (transcribed) strand is repaired at such a high efficiency as to account for all ofthe gene-specific repair, whereas the coding (nontranscribed) strand is repaired 'at essentially the same rate as the rest of the genome (4, 5). Recently, we have developed an in vitro system (6, 7) capable of gene-and strand-specific repair and we have partially puified an Escherichia coli protein that confers strand specificity onto the E. coli nucleotide excision repair enzyme, (A)BC excinuclease. In this communication we describe the purification of the "transcription-repair coupling factor"' (TRCF) to nearhomogeneity and the preliminary identification of the coupling factor as the mfd gene product.MFD (mutation'frequency decline) is operationally defined as the rapid and irreversible decrease in the frequency of certain damage-induced suppressor mutations that occurs when protein synthesis is transiently inhibited immediately after irradiation (8)(9)(10)(11)(12) from' E. coli B/r and its mfd derivative were tested for strand-specific repair in vitro. We found that E. coli B/r, like E. coli K-12, was capable of strand-specific repair. In contrast, E. coli B/r mfd-extract was totally deficient in strand-specific repair. When we added the purified TRCF to the mutant cell extract it restoredthe strand-specific repair to the wild-type level. The most likely explanation of our data is that nfd encodes the TRCF. MATERIALS AND METHODS Cdls and.. E. coli K-12 derivatives AB1157 (wild type) and AB1886 (uvrA-) were used for making extractsfor routine repair synthesis assay and for purification of the TRCE, respectively. E. coli B/r derivative WU3610 (which is Leu-and Tyr-because of UAG and UAA mutations) and its derivative' WU3610-45 (mfd-1) are the strains that have frequently been used in studies on MFD (11). The plasmid pDR3274 (19) contains the uvrC gene under the strong tac promoter. Transcription from this promoter can be inhibited by rifampicin (Rif) ...