It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM), exhibits several characteristics distinct from mutations that occur within the course of replication: i) following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min) than replication dependent mutations (t ½ ≈ 80–100 min) ii) NERiM specifically requires DNA Pol IV in addition to Pol V iii) NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure) of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication.
Many endogenous and exogenous chemicals as well as radiations covalently damage DNA leading to major perturbations of DNA metabolism such as in replication and transcription. Cells have evolved sophisticated mechanisms to deal with DNA damage, collectively referred to as DNA Repair mechanisms. In principle, DNA Repair pathways refer to processes that effectively remove DNA lesions. Most repair pathways rely on the double-stranded nature of DNA that allows a damaged strand to be corrected by using the information present in the complementary strand. For instance Nucleotide Excision Repair (NER) uses dedicated enzymes that recognize damaged DNA, incise the damaged strand and remove a short oligonucleotide encompassing the damage. Regulation of the NER pathway requires high specificity in lesion recognition by means of molecular matchmaking and kinetic proofreading and proper coordination with the DNA Damage checkpoint response [1–4]. The resulting single-stranded DNA gap is accurately filled-in by a DNA polymerase and sealed by a ligase. This “cut and patch” repair mechanism has a huge effect on protecting cells against genotoxic compounds, indeed an NER-proficient strain is resistant to a UV dose that is almost 50-fold higher compared to an NER-deficient strain [5,6]. Nevertheless, a few lesions may escape repair and will be present at the time of DNA replication, thus perturbing replication fork progression. In that case, cells implement lesion tolerance pathways that are not genuine repair pathways since the lesion is bypassed without being removed [7]. A common lesion tolerance strategy, named Translesion Synthesis (TLS), involves specialized DNA polymerases that are able to copy damaged templates. TLS occurs either directly at the fork or behind the fork during a gap-filling process [8]. Due to the low fidelity of the TLS polymerases, this process is intrinsically error-prone. In E. coli, Pol V, encoded by the umuDC locus, plays a prominent role in Translesion Synthesis (TLS) and mutagenesis [9,10]. Indeed, essentially all UV-induced mutagenesis is abolished when umuDC is inactivated. The primary role of Pol V in UV-induced mutagenesis is due to its unique property to mediate the specific insertion steps across the major UV lesions [11,12]. The early-time responses of E. coli cells to UV irradiation have been monitored by the kinetics of dNTP incorporation into newly synthesized DNA [13–16]. A common conclusion from these investigations is that, after irradiation of a wild type strain, following an initial abrupt drop, the rate of synthesis increases to progressively reach its normal speed over a period 40–50’ post irradiation. Compared to a wild type strain, inactivation of the TLS polymerases only modestly affects the recovery process [13,14]. This observation can be interpreted as follows: when the replication fork encounters a lesion the fork does not stop permanently. Indeed, when a DNA polymerase encounters a replication-blocking lesion in the leading strand, the replicative helicase continues unwinding the two strands but at a highly reduced rate due to its uncoupling from the DNA polymerase [17,18] before downstream re-priming eventually occurs [19]. Downstream re-priming events create gaps that are subsequently repaired most often by recombination with the sister chromatid or more rarely by TLS [20]. The observed reduction in the rate of DNA synthesis may thus reflect the reduced fork speed due to the uncoupling / re-priming process [5,6, 21] rather than indicating a complete stop in replication fork progression as often suggested [7,22]. During the ≈ 50’ period, NER removes most of the lesion genome-wide to permit recovery of full synthesis rate [14–16,23]. Not surprisingly, in a uvrA strain the rate of synthesis post UV irradiation is strongly affected [14]. The notion that mutagenesis may be associated, at least in part, with NER has been suggested more than 40 years ago by Nishioka and Doudney who reported that UV mutagenesis coincides with the loss of photoreversibility of UV lesions within a 20 min post UV irradiation period in a wild-type but not in a uvrA strain [24,25]. These data were interpreted as evidence for a NER-dependent pathway that induces mutations in a wild-type strain at early time points following UV irradiation. Bridges extended this observation by showing that the NER-dependent mutation pathway was recA+ dependent [26]. Moreover, evidence for NER-dependent UV mutagenesis was described in vitro in E. coli crude extracts [27,28]. In NER-proficient S. cerevisiae, it was shown that mutations can occur prior to the first post-UV replication cycle [29] [30] while mutations are not fixed prior the first post-UV irradiation replication cycle in NER-deficient yeast cells, [31]. Studies on the repair of clustered lesions in double-stranded DNA have mostly been restricted to lesions induced by radiations such as Uracil residues, abasic sites or 8-oxo-G which are typical substrates for Base Excision Repair pathways (for a review see [32]). The concept that closely spaced UV lesions are a potential source of mutations has been documented and discussed in bacteria [33,34] and in yeast where it was shown that mutations accumulate as a function of the square UV dose in wild-type cells but not in NER-deficient cells [30]. More recently, the occurrence of UV-induced mutations in non-proliferating yeast cells prior to replication was demonstrated [35]. While this class of mutations is characterized by the occurrence of the mutation in both DNA strands prior to replication in a NER-dependent way, the data did not allow distinguish between a linear or a quadratic dose-response curve. Despite all these data, the precise mechanistic and genetic links between NER and mutagenesis have largely remained unexplored. In the present paper, we show that in NER-proficient strains, a significant fraction of UV-induced mutagenesis occurs within the time frame of active NER, i. e. when replication is shutdown. Interestingly, these early time-point mutations disappear in NER-deficient strains. Thus, in proliferating cells, UV-induced mutations fall in two classes, mutations induced during replication (RiM) and NER-induced mutagenesis (NERiM), respectively. We also show that mutations induced in a NER-dependent way accumulate with the square of the UV dose suggesting a “two-hit mechanism”. Normally, the NER machinery excises lesions as small damage-containing oligonucleotides and leaves a short gap that is filled-in; however, if following an initial NER incision event, another lesion resides in close proximity in the complementary strand, the second lesion may become exposed within the initial NER gap forming a so-called opposing lesion structure. The second lesion may be converted into a point mutation during filling-in of the NER associated gap. The mutagenic process associated with NER not only requires Pol V, but also DNA Pol IV and to some extent of Pol II. Additionally, we would like to stress that when two lesions are located in close proximity in the same strand, processing of one of them by NER may also trigger gap enlargement and subsequent requirement for processing by Pol IV/Pol II. For sake of simplicity, we will essentially discuss the situation were the closely spaced lesions are located in opposite strands. Our data are consistent with NERiM playing a significant role in UV mutagenesis in proliferating cells; indeed, even at relatively low levels of irradiation (40J/m2), NERiM accounts for 1/3 of UV-induced mutagenesis. In addition we show that the role of NERiM becomes predominant in stationary phase bacteria suggesting an elegant way to generate genetic diversity in non-replicating microbes and perhaps in post-mitotic metazoan cells.
As discussed in the introduction, following UV irradiation, bacterial cells temporarily stop replication to allow removal of most lesions by NER before resuming replication [14–16,23]. We thought to exploit this feature as a way to differentiate mutations that are fixed during repair from those that arise during replication by analyzing the induction of mutations in various strains as a function of time after UV. We thus compared the kinetics of rifR mutation fixation in various strains following a single UV dose. The UV dose was chosen so as to affect survival in the various strains to a similar extent, namely ≈10% (wild-type: 100 J/m2, dinBpolB: 35 J/m2, uvrA: 3 J/m2). While rifR mutations accumulate rapidly in the wild-type strain (t1/2 ≈ 30 min after irradiation), the kinetics of appearance of mutations in the uvrA and in the dinBpolB strains are very significantly delayed (t1/2 ≈ 80–100 min after irradiation) (Fig 2A). Mutations fixed during replication appear with a delayed kinetics compared to NER-associated mutations. The plateau reached in the uvrA and dinBpolB strains corresponds to ≈ 25–30% and 15–20% of the level reached in the wild-type strain, respectively (see also Fig 1E at 10% of survival). As indicated above, for a wild-type strain a dose of 100J/m2 corresponds to a similar level of survival (≈10%) as a dose of 35J/m2 administered to a dinBpolB strain (Fig 1C). According to our mathematical model (see below), the level of RiM as deduced from the dinBpolB strain at 35J/m2 corresponds to only ≈15% of the total level of mutagenesis induced at 100J/m2 in the wild type strain. This is compatible with the observation that in the wild-type strain most of the induced mutagenesis occurs rapidly via NERiM and that only a small additional increase in mutagenesis appears at longer time points (corresponding to ≈15% of RiM). To further establish this hypothesis we conducted UV-induced mutagenesis experiments in a thermo-sensitive dnaB helicase mutant. These experiments were conducted at a UV dose of 90 J/m2 corresponding to about 10% of survival (Fig 2B). In a dnaBts strain, known to be a quick-stop replication mutant [45,46], rifR mutants are induced at similar kinetics at both permissive (35°C) and non-permissive (42°C) temperature, implying that most mutations are fixed in a non-replicative manner (Fig 2B). How may UV-induced mutagenesis be triggered during NER? We envision two formal possibilities i) the “faulty NER” model: at low frequency, the NER machinery makes a “mistake” and incises the non-damaged strand thus generating a lesion-containing gap or ii) the “opposing lesion structure” model: a proper NER-mediated repair event occurs at a location where a second lesion resides in close proximity in the complementary strand. In both models, the gap resulting from NER incision contains a lesion that may lead to a mutation during subsequent gap filling. To distinguish between the two models, we analyzed the shape of the induced mutagenesis curve as a function of UV doses. In the wild-type strain, mutation frequency data exhibit a near-quadratic behavior with respect to UV dose (Fig 3A and 3B) while the relationship is linear in both dinBpolB (Fig 3C) and uvrA (Fig 3D) strains. The quadratic response in the wild-type strain suggests that NERiM results from the processing of opposing lesions (see below). In contrast, the linear relationship of mutation frequency with dose in the dinBpolB and uvrA strains indicates a single hit model as expected when mutations are fixed during replication (RiM). We propose the following working model for NERiM (Fig 4A): normally, NER proceeds via excision of a short 12–13 nucleotide-long tract that contains a damaged nucleotide, followed by repair synthesis of the short gap by DNA Pol I. This process is error-free and leads to a spectacular increase in cell survival (Fig 1C). At rare occasions a second lesion will be located in the opposite strand either within the initial incision gap or in close proximity, near the gap junctions. When the second lesion is located within the initial gap, a gap enlargement step may be necessary to allow a RecA filament to assemble downstream from the lesion in order to activate Pol V [47]. If the second lesion is located near the initial gap junction, it may become part of the gap following a gap enlargement step triggered by the distortion induced by the second lesion at the double-stranded / single-stranded junction via exonuclease (or helicase) processing. The resulting structure will be referred to as “opposing lesion structure”. Closely spaced photoproducts in complementary strands occur at high (but physiologically relevant) doses [48]. Published data suggest that opposing UV dimers are substrates for NER incision. Indeed, opposing cyclobutane dimers within the 5’-TTAA sequence are properly incised by UvrABC, in one or the other strand, without double strand break formation [49]. The frequency of opposing lesion structures increases with the square of the UV dose. The present model posits that repair of the opposing lesion structure requires both Pol V at the TLS step across the lesion and Pol IV / Pol II for yet unknown reasons (see below the § about Damaged Primer Elongation) (Fig 4A). However, it does not exclude the potential participation of Pol III in the gap-filling reaction as previously suggested by Bridges and coworkers [50]. This model predicts similar UV-sensitivities for dinBpolB and umuDC strains, an expectation not met experimentally as a umuDC strain is found to be less UV-sensitive than a dinBpolB strain (Fig 1C). The higher UV sensitivity of polBdinB compared to umuDC could be accounted for by the existence of NER-induced gaps that require processing by Pol IV/Pol II but not by Pol V. Such gaps may contain a specific UV lesion that can be bypassed by Pol IV/Pol II without need of Pol V (as discussed above: the putative lesion at position 526). Alternatively, when two lesions are in close proximity in the same strand, incision at one of them may generate a lesion-containing primer that requires Pol IV / Pol II for its extension (see below) but not necessarily Pol V as the gap may not contain a lesion. Such events account for greater UV sensitivity of dinBpolB compared to umuDC strains. The precise role of Pol IV (and to a lesser extent of Pol II) during the completion of the gap-filling reaction during NERiM remains a puzzle for which we offer some genetic and biochemical hints (see below). To better define the requirement for Pol IV, we wondered whether its role in NERiM requires binding to the beta-clamp, the replication processivity factor [51]. For this purpose we used a mutant dinB gene carrying a five amino acid C-terminal deletion of the consensus beta-clamp binding motif [52]. Compared to a plasmid expressing dinB+, a plasmid expressing the dinBΔ5 mutant only partially complemented a dinBpolB strain (Fig 1A), suggesting that the interaction of Pol IV with the beta-clamp is important for its function in NERiM. Interestingly, Pol kappa, the eukaryotic ortholog of Pol IV was shown to be involved in NER [53]. The model proposed could explain the finding that mouse cells lacking Pol kappa are defective at excising lesions from DNA and filling NER gaps [54]. In order to estimate the mutation frequencies induced by UV lesions during NER (NERiM) or replication (RiM) as a function of UV dose (D), we have generated the following mathematical model. RiM and NERiM will be calculated by multiplying the number of lesions subject to either replication or NER by the average rate of conversion of a lesion into a mutation in the rif assay. The following parameters have been used: We wondered whether the NER-induced mutation pathway described above and established within the frame of a chromosomal mutation assay (rifR) using UV lesions as the mutagen could be generalized to another mutagen and another assay. For this purpose we implemented a plasmid-borne reversion assay using as a mutagen oxidative lesions induced by the in vitro treatment of plasmid DNA with methylene blue plus visible light (MB+light) and its subsequent introduction in bacterial cells for mutagenesis monitoring. This mutation assay involves the reversion of a tetracycline sensitive to a tetracycline resistant allele carried by a pBR322-derived plasmid [58]. Plasmid DNA is randomly damaged in vitro with (MB+ light) and introduced into bacteria by transformation. The assay was shown to be highly specific for monitoring only true -2 frameshift revertants to tetracycline resistance [59]. This assay is highly sensitive as the level of induced mutagenesis is two to three orders of magnitude above the background level reached with untreated control plasmid [60]. Cells are plated on tetracycline and ampicillin plates to determine the TetS-> TetR mutation frequency. The (MB+light) treatment, that induces a variety of oxidative lesions, was previously shown to trigger -2 frameshifts in an SOS-dependent way [60]; although the chemical nature of the culprit lesion is unknown, it was shown not to be 8-oxo-dG [60]. Introduction into SOS-induced wild-type cells of plasmid DNA treated with (MB+light) robustly increases the -2 frameshift mutation frequency by two to three orders of magnitude above untreated control plasmid (Fig 5A and 5B). The mutagenic response is reduced ≈5-fold when the (MB+light) treated plasmid is introduced into either uvrA, dinBpolB or uvrAdinBpolB strains (Fig 5B). In a umuDC strain the induced-mutation frequency is fully abrogated [61]. These data, obtained within the frame of a plasmid-borne mutation assay, fully recapitulate the key genetic requirements of NERiM as established above with the chromosomal rifR assay. It thus further generalizes NERiM to different mutagenic treatments and to all types of point mutations, base substitutions and frameshift mutations. Moreover, the plasmid assay could be implemented in all strains at the same lesion density, thus validating the NERiM pathway established at doses leading to equal survival but with different lesion densities in the chromosomal context (see further discussion in Conclusion §). Next, we wondered about the biological significance of NERiM, a mutation pathway that is coupled to NER instead of being linked to replication. A NER-dependent pathway appears to be advantageous to stationary phase cells in order to adapt to genotoxic conditions. To investigate directly this possibility we UV-irradiated stationary-phase bacteria contained in 1 to 3 day-old E. coli colonies (doses 200–400 J/m2) and further incubated the colonies for periods ranging between 1 to 3 days. At the end of the incubation period, the level of rifR mutants was determined by suspending individual colonies in liquid medium and plating adequate dilutions on LB or LB + rifampicin containing plates. In wild-type bacteria, we observe a 30 to 50-fold increase in rifR mutagenesis in irradiated compared to non-irradiated control colonies. Strikingly, induced mutagenesis in these stationary phase E. coli cells was entirely dependent upon a functional excision repair system (i. e. absence of induced mutagenesis in a uvrA strain) suggesting that mutagenesis arose during NER (Fig 6A). In order to determine which DNA polymerase was involved in the generation of these UV-induced mutations, we examined strains defective in each of the specialized DNA polymerases, Pol II, Pol IV and Pol V. In addition to the expected involvement of Pol V, UV-induced mutagenesis in stationary phase cells was totally dependent upon a functional Pol IV gene, while Pol II had essentially no effect. Thus the genetic control of stationary phase mutagenesis induced by UV irradiation is similar to NERiM except for the involvement of Pol II, an observation that remains unexplained. We also found that UV-induced mutagenesis in stationary phase cells is suppressed in a strain that cannot induce its SOS system (lexA (Ind) - strain) (Fig 6B). It has also been reported that spontaneous mutagenesis in resting bacterial populations is controlled by the SOS-response in a cAMP-dependent way [62]. Using an F plasmid based reversion assay, it was shown that spontaneous -1 frameshift mutations in short runs depend upon Pol IV and recombination proteins in stationary phase cells [63]. Detection of lesions in stationary phase may be initiated during transcription by the Transcription Coupled Repair factor Mfd as suggested by a recent study on stationary phase mutagenesis in B. subtilis [64]. In B. subtilis it was also found that stationary-phase mutagenesis involves the yqjH gene that encodes a Pol IV homolog [65]. Our present working model for NER-induced mutations involving opposing lesions (Fig 4A) raises two major questions: how are the extended gaps formed and how are they subsequently filled-in? We hypothesize the involvement of an exonuclease activity that will use as its substrate a normal NER gap formed at opposing lesion structures (as defined above, Fig 4A). Similarly, in S. cerevisiae it was shown that the occasional processing of normal NER incision intermediates by Exo1 leads to extended gaps that are essential in checkpoint activation in non-dividing cells [66,67]. The rare occurrence of large NER gaps has previously been described in E. coli [68,69]. Indeed, large NER gaps (≈1500 bases) were found at about 1% the frequency of the normal NER gaps for UV doses in the 60J/m2 range [68,69]. The enzymatic activities involved in gap enlargement in E. coli are unknown. E. coli possesses numerous exonucleases preventing us to unequivocally identify the responsible exonuclease (s). Degradation by exonucleases of DNA templates containing chemically modified nucleotides has revealed that the degradation of lesion-containing templates is blocked at or near the lesion sites [70,71]. If this is the case, the subsequent gap-filling reaction requires a DNA polymerase able to extend a primer containing a lesion in the vicinity of its 3’-end (Fig 4A). In order to support such a model, the ability of E. coli’s DNA polymerases to extend damaged primer extremities in vitro was analysed. Primers containing either a TT-CPD or a T (6–4) T photoproduct at their 3’-end (S3 Fig) were annealed to a single-stranded circular DNA substrate (S4 Fig). These primer / templates were subsequently incubated in the presence of the β-clamp, the clamp loader, RecA and SSB proteins before addition of a given DNA polymerase [12]. For sake of comparison, an equivalent amount of DNA pol units of Pol I, II, III*, IV and V were added to these primer template substrates although this does not reflect the respective amounts of the different DNA polymerases in E. coli cells. Following a 30 minutes incubation period, replication products were analysed by PAGE. The data show that for the TT-CPD containing primer, Pol IV (and Pol II) show significantly greater extension capacities compared to Pol I and Pol V, while Pol III* is intermediate (S4 Fig). With respect to the T (6–4) T bearing primer, Pol II exhibits the greatest extension potential (S4 Fig). The lack of extension activity by Pol I is in good agreement with our in vivo data showing that NERiM is fully proficient in the polA1 strain (Fig 1B). In good agreement with genetic data, Damaged Primer Extension of TT-CPD and T (6–4) T lesions by Pol IV and Pol II, rely on the presence of the β-clamp (S5 Fig) (Fig 1A). These data support the idea that Pol IV / Pol II may be involved in NERiM by virtue of their capacity to extend primers that contain lesions at their 3’-extremity (S6 Fig).
In this paper, we report the surprising finding that in addition to the well-known properties of Nucleotide Excision Repair (NER) in efficiently repairing a large number of DNA lesions, NER entails a mutagenic sub-pathway. Our data suggest that closely spaced lesions are processed by NER into a toxic DNA intermediate, i. e. a gap containing a lesion, that leads either to mutagenesis during its repair or to cell death in the absence of repair. The paper describes a new pathway for the generation of mutations in stationary phase bacteria or quiescent cells; it also provides an additional role for Pol IV, the most widely distributed specialized DNA polymerase in all forms of life.
mutagenesis
biochemistry
mutation
dna damage
point mutation
dna polymerase
mutant strains
polymerases
dna replication
nucleic acids
proteins
genetics
biology and life sciences
dna
dna-binding proteins
frameshift mutation
Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli