Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli

Koffel-Schwartz, Nicole; Verdier, Jean‐Michel; Bichara, Marc; Freund, Anne-Marie; Daune, Michel; Fuchs, Robert P. P.

doi:10.1016/0022-2836(84)90056-1

Cited by 179 publications

(177 citation statements)

References 27 publications

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“…We observed that DnaE was able to bypass N2-acetylaminofluorene guanine adduct (G-AAF) located in three different sequence contexts (Fig. 7A) (40,41). Bypass of the 3G 1 -AAF substrate was inefficient (Fig.…”

Section: Resultsmentioning

confidence: 98%

Involvement of DnaE, the Second Replicative DNA Polymerase from Bacillus subtilis, in DNA Mutagenesis

Bécherel¹,

d’Alençon²,

Canceill³

et al. 2004

Journal of Biological Chemistry

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In a large group of organisms including low G ؉ C bacteria and eukaryotic cells, DNA synthesis at the replication fork strictly requires two distinct replicative DNA polymerases. These are designated pol C and DnaE in Bacillus subtilis. We recently proposed that DnaE might be preferentially involved in lagging strand synthesis, whereas pol C would mainly carry out leading strand synthesis. The biochemical analysis of DnaE reported here is consistent with its postulated function, as it is a highly potent enzyme, replicating as fast as 240 nucleotides/s, and stalling for more than 30 s when encountering annealed 5-DNA end. DnaE is devoid of 3 3 5-proofreading exonuclease activity and has a low processivity (1-75 nucleotides), suggesting that it requires additional factors to fulfill its role in replication. Interestingly, we found that (i) DnaE is SOS-inducible; (ii) variation in DnaE or pol C concentration has no effect on spontaneous mutagenesis; (iii) depletion of pol C or DnaE prevents UV-induced mutagenesis; and (iv) purified DnaE has a rather relaxed active site as it can bypass lesions that generally block other replicative polymerases. These results suggest that DnaE and possibly pol C have a function in DNA repair/mutagenesis, in addition to their role in DNA replication.In all living organisms, DNA replication is carried out by a functionally highly conserved protein complex. Genetic and biochemical data have shown that this complex, called DNA polymerase holoenzyme, contains two copies of an essential replicative DNA polymerase in Escherichia coli, T4 and T7 phages, and SV40 (reviewed in Refs. 1-6). In contrast, replication requires two different polymerases in bacteria Bacillus subtilis and Staphylococcus aureus (pol 1 C and DnaE, C family (7, 8)) and in eukaryotes including Saccharomyces cerevisiae, Xenopus, and human (pol ␦ and pol ⑀, B family; reviewed in Refs. 5 and 9 -11). Thus, holoenzyme of these organisms might be more complex, containing two different polymerases instead of two copies of a single polymerase. This higher level of complexity would hold true for many organisms as follows: (i) systematic sequencing of bacterial genomes (more than 100 completed to date) revealed that ϳ50% carry at least two copies of dnaE or contain dnaE and polC (no genome containing only polC has been detected so far), and (ii) pol ␦ and pol ⑀ seem to be ubiquitous in eukaryotes. It is well established that pol C in bacteria and pol ␦ in eukaryotes are required at the replication fork (5, 9 -15). On the other hand, the specific roles of DnaE and pol ⑀ during replication are still not known. In B. subtilis, it was reported that the purified DnaE protein has a DNA polymerase activity devoid of proofreading activity and presents a high affinity for dNTP (14,16). Genetic and cytological data as well as in vivo assays of radioactive precursor incorporation have shown that DnaE, like pol C, is essential for the elongation phase of replication and is associated with the replication factory at mid-cell (7). Moreover, stu...

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Section: Resultsmentioning

confidence: 98%

Involvement of DnaE, the Second Replicative DNA Polymerase from Bacillus subtilis, in DNA Mutagenesis

Bécherel¹,

d’Alençon²,

Canceill³

et al. 2004

Journal of Biological Chemistry

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“…These adducts have been studied extensively in terms of the structural deformation that they induce in double-stranded DNA (insertion-denaturation model for AAF adducts and outside-binding model for AF adducts) (25)(26)(27)(28) and their effect on DNA metabolism. AAF adducts are strong frameshift mutagens in vivo, inducing both -1 and -2 frameshift mutations in hotspot sequences (8,9), whereas AF adducts induce primarily base-pair substitutions, especially G --T transversions (29). AAF adducts strongly block DNA synthesis by several purified DNA polymerases in vitro, whereas the same polymerases are able to synthesize through the AF adduct after a brief pause (1,6,22).…”

Section: Resultsmentioning

confidence: 99%

“…For example, there is no in vitro trans-lesion synthesis (TLS) by most DNA polymerases through N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) (1), an adduct formed at the C8 position of guanine by the rodent hepatocarcinogen N-2-acetylaminofluorene (AAF) (7). However, in vivo doublestranded plasmids containing AAF adducts can replicate in excision-repair deficient hosts in an error-free or error-prone (mutagenic) manner (8)(9)(10)(11). These observations confirm the fact that there are efficient mechanisms that rescue a blocked replication fork in vivo (12).…”

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confidence: 99%

Cellular strategies for accommodating replication-hindering adducts in DNA: control by the SOS response in Escherichia coli.

Koffel-Schwartz¹,

Coin²,

Veaute³

et al. 1996

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

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The replication of double-stranded plasmids containing a single adduct was analyzed in vivo by means of a sequence heterology that marks the two DNA strands. The single adduct was located within the sequence heterology, making it possible to distinguish trans-lesion synthesis (TLS) events from damage avoidance events in which replication did not proceed through the lesion. When the SOS system of the host bacteria is not induced, the C8-guanine adduct formed by the carcinogen N-2-acetylaminofluorene (AAF) yields less than 1% of TLS events, showing that replication does not readily proceed through the lesion. In contrast, the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene yields -70% of TLS events under both SOS-induced and uninduced conditions. These results for TLS in vivo are in good agreement with the observation that AAF blocks DNA replication in vitro, whereas aminofluorene does so only weakly. Induction of the SOS response causes an increase in TLS events through the AAF adduct ("13%). The increase in TLS is accompanied by a proportional increase in the frequency of AAF-induced frameshift mutations. However, the polymerase frameshift error rate per TLS event was essentially constant throughout the SOS response. In an SOS-induced AumuD/C strain, both TLS events and mutagenesis are totally abolished even though there is no decrease in plasmid survival. Error-free replication evidently proceeds efficiently by means of the damage avoidance pathway. We conclude that SOS mutagenesis results from increased TLS rather than from an increased frameshift error rate of the polymerase.Most mutagens and carcinogens react with the bases of DNA by forming covalent adducts that interfere with DNA metabolism if left unrepaired. Repair pathways remove these lesions: excision repair removes damaged bases or nucleotides prior to replication and post-replication repair fills in gaps formed in newly synthesized DNA strands when damaged DNA is replicated. These two major repair pathways are believed to be essentially error-free and to contribute about equally to cell survival. The effect of DNA adducts on replication has been analyzed in in vitro assays using damaged single-stranded DNA templates and purified DNA polymerases (1-6). Studies of templates containing a single adduct have shown that some adducts are absolute blocks for in vitro DNA synthesis. For example, there is no in vitro trans-lesion synthesis (TLS) by most DNA polymerases through N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) (1), an adduct formed at the C8 position of guanine by the rodent hepatocarcinogen N-2-acetylaminofluorene (AAF) (7). However, in vivo doublestranded plasmids containing AAF adducts can replicate in excision-repair deficient hosts in an error-free or error-prone (mutagenic) manner (8-11). These observations confirm the fact that there are efficient mechanisms that rescue a blocked replication fork in vivo (12). Two such pathways are TLS involving a modified replisome and damage avoidance (DA) mechanisms that use ...

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“…In contrast, when Pol II is used in the complete TLS reaction, involving both insertion and extension steps, both Ϫ1 and Ϫ2 TLS products are observed. That Ϫ1 frameshift mutations are not found at NarI sites in vivo (29,39) suggests that the reaction leading to the Ϫ1 TLS product in vitro is not biologically relevant. Therefore, most likely, in vivo, Pol II is involved not in the insertion step but only in the extension step(s).…”

Section: Insertionmentioning

confidence: 99%

Mechanism of DNA polymerase II-mediated frameshift mutagenesis

Bécherel

Fuchs

2001

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

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Escherichia coli possesses three SOS-inducible DNA polymerases (Pol II, IV, and V) that were recently found to participate in translesion synthesis and mutagenesis. Involvement of these polymerases appears to depend on the nature of the lesion and its local sequence context, as illustrated by the bypass of a single N-2-acetylaminofluorene adduct within the NarI mutation hot spot. Indeed, error-free bypass requires Pol V (umuDC), whereas mutagenic (؊2 frameshift) bypass depends on Pol II (polB). In this paper, we show that purified DNA Pol II is able in vitro to generate the ؊2 frameshift bypass product observed in vivo at the NarI sites. Although the ⌬polB strain is completely defective in this mutation pathway, introduction of the polB gene on a low copy number plasmid restores the ؊2 frameshift pathway. In fact, modification of the relative copy number of polB versus umuDC genes results in a corresponding modification in the use of the frameshift versus error-free translesion pathways, suggesting a direct competition between Pol II and V for the bypass of the same lesion. Whether such a polymerase competition model for translesion synthesis will prove to be generally applicable remains to be confirmed. NarI mutation hot spot ͉ translesion synthesis ͉ slippage mutagenesis ͉ N-2-acetylaminofluorene ͉ umuDC (Pol V) P oint mutations are formed during DNA replication either as genuine replication errors or as a consequence of the presence of damage in the parental DNA. By changing the chemical structure of the bases, damaging agents are often effective blocks to the progression of replicative DNA polymerases. It has now become clear that these blocks are overcome by specialized enzymes (translesional DNA polymerases) that can read through damaged bases in a process known as translesion synthesis (TLS) (1-5). Because of the presence of the lesion, this process is inevitably less accurate than normal replication and will thus trigger increased mutation rates in the newly synthesized strand opposite the damaged base.Recently it was shown that in Escherichia coli, all three SOS-inducible DNA polymerases, Pol II (polB), Pol IV (dinB), and Pol V (umuDC), can be involved in TLS, depending on the nature of the DNA damage and its sequence context (6). We have identified an intriguing situation where the bypass of a given lesion, N-2-acetylaminofluorene (AAF), located within a frameshift mutation hot spot, the NarI site, is mediated by two genetically distinct pathways. Indeed, in that sequence context, the bypass of an AAF guanine adduct requires Pol V for nonslipped elongation, yielding error-free TLS but Pol II for slipped elongation, thus producing Ϫ2 frameshift TLS (6). The nonslipped TLS pathway obeys the rules previously described for base substitution mutagenesis induced by UV light or abasic sites, i.e., Pol V and RecA* dependence (for a recent review, see ref. 7). In contrast, the involvement of DNA Pol II in the slipped Ϫ2 frameshift pathway is more intriguing. Although a series of phenotypes related to DNA repair...

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Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli

Cited by 179 publications

References 27 publications

Involvement of DnaE, the Second Replicative DNA Polymerase from Bacillus subtilis, in DNA Mutagenesis

Involvement of DnaE, the Second Replicative DNA Polymerase from Bacillus subtilis, in DNA Mutagenesis

Cellular strategies for accommodating replication-hindering adducts in DNA: control by the SOS response in Escherichia coli.

Mechanism of DNA polymerase II-mediated frameshift mutagenesis

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