Nucleotide excision repair is universally mutagenic and transcription-associated

Nucleotide excision repair (NER) is a highly conserved mechanism that removes lesions from DNA. This process has been studied for decades, however, almost all of the work on NER was performed in the presence of exogenous DNA damage. Under these conditions, NER is anti-mutagenic in bacteria. Here, we describe our findings on the role of NER in mutagenesis under endogenous conditions. Counter to dogma, we find that NER is actually pro-mutagenic. Our data suggest a hand-off mechanism between two different types of DNA polymerases that explains the mutagenic nature of NER. Additionally, NER is thought to occur in two different ways; 1) in a transcription-coupled manner where it plays a role in removing lesions that block RNA polymerase, and 2) in a process known as global genome NER, which is independent of transcription. Counter to the classical view, our genetic analyses of the relationship between NER and the RNA polymerase interacting DNA translocase, and evolvability factor, Mfd, indicate that most likely all NER is associated with transcription. Lastly, we show that NER is pro-mutagenic because of endogenous oxidative damage. Altogether, our data strongly suggest that oxidative damage induces a mutagenic NER mechanism, which then accelerates evolution across divergent bacterial species.


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Nucleotide excision repair (NER) is a highly conserved mechanism that removes lesions from DNA. This 23 process has been studied for decades, however, almost all of the work on NER was performed in the 24 presence of exogenous DNA damage. Under these conditions, NER is anti-mutagenic in bacteria. Here, 25 we describe our findings on the role of NER in mutagenesis under endogenous conditions. Counter to 26 dogma, we find that NER is actually pro-mutagenic. Our data suggest a hand-off mechanism between 27 two different types of DNA polymerases that explains the mutagenic nature of NER. Additionally, NER is 28 thought to occur in two different ways; 1) in a transcription-coupled manner where it plays a role in 29 removing lesions that block RNA polymerase, and 2) in a process known as global genome NER, which is 30 independent of transcription. Counter to the classical view, our genetic analyses of the relationship 31 between NER and the RNA polymerase interacting DNA translocase, and evolvability factor, Mfd, 32 indicate that most likely all NER is associated with transcription. Lastly, we show that NER is pro-33 mutagenic because of endogenous oxidative damage. Altogether, our data strongly suggest that 34 oxidative damage induces a mutagenic NER mechanism, which then accelerates evolution across 35 divergent bacterial species. 36 37 38 Main text 39 Mutations drive evolution. They provide the necessary genetic diversity that natural selection can 40 then use to help organisms adapt to new environments. Even though mutations are mostly 41 deleterious, and that lower mutation rates are generally accepted to be beneficial, all organisms 42 have a baseline mutation rate that allows them to evolve 1 . However, which endogenous 43 mechanisms most commonly lead to mutations and drive evolution remain poorly understood. 44 We recently determined that the bacterial RNA polymerase interacting protein Mfd, which is 45 involved in a sub-pathway of the DNA repair pathway nucleotide excision repair (NER), 46 promotes mutations and leads to rapid antibiotic resistance development across highly divergent 47 bacteria 2 . This function depended on Mfd's interaction with the NER protein UvrA. These 48 findings suggested that NER in general might be a mechanism that drives spontaneous 49 mutagenesis in bacteria, potentially even in the absence of transcription. However, this was a 50 hypothesis that would go against dogma. The dogmatic view of NER and its role in reducing 51 mutagenesis was based on a series of studies that utilized artificial DNA damaging conditions. 52 Most in vivo studies on NER to date have been performed in the presence of exogenous DNA 53 damage such as UV light. These studies led to the conclusion that NER is an anti-mutagenic 54 DNA repair pathwayfa 3,4 . However, whether this is true in an endogenous context, in 55 unperturbed cells, in the absence of artificial DNA damaging conditions, has not been 56 investigated.

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Traditionally, bacterial NER has been thought to consist of two sub pathways that differ only on all NER is transcription coupled, although these conclusions remain somewhat controversial 7-10 . 68 Here we show that the core NER factors UvrABC promote spontaneous mutagenesis, across 69 highly divergent bacterial species. We also show that a replicative polymerase, and two Y-family 70 polymerases are responsible for the observed NER-dependent increase in mutagenesis. Critically, 71 our genetic analyses show that all three polymerases function in the same pathway as the NER 72 proteins and Mfd. These findings strongly suggest that bacterial NER is a pro-mutagenic repair 73 pathway that is universally coupled to transcription, and that this is due to the usage of error 74 prone polymerases. Last, we present data that the underlying cause of this process is endogenous 75 oxidative stress and that in general, oxidative damage drives NER mutagenesis and rapid 76 evolution in bacteria. 78 We and others have previously shown that, in the absence of exogenous DNA damage, the 79 bacterial transcription-coupled repair (TCR) protein Mfd promotes mutagenesis across many 80 different bacterial species 2,11,12 . In addition, we previously showed that this pro-mutagenic effect 81 depends on the interaction of Mfd with the RNA polymerase (RNAP) and the NER protein 82 UvrA 2 . This brings up the possibility that NER, which is thought to promote genome stability 83 and reduce mutagenesis 3,4 , is pro-mutagenic in the absence of exogenous DNA damage, in the 84 endogenous context. 85 We first tested this hypothesis by measuring mutation rates using the Luria-Delbruck fluctuation 86 assays 13 in wild-type Bacillus subtilis strains and isogenic strains that lack each of the NER 87 proteins: UvrA, UvrB and UvrC. We performed these experiments in the absence of exogenous 88 DNA damage. Strikingly, we observed a 50-75% decrease in the mutation rates in NER deficient 89 strains (Fig. 1A). To determine whether the pro-mutagenic effects of NER were due to TCR and/or GG-NER, we 99 built double mutants that lacked Mfd and the Uvr proteins. If NER is mutagenic due to only 100 TCR, then we expected that the double mutants lacking Mfd and NER proteins would have an 101 epistatic relationship, and that the mutation rates of the double mutants would be similar to the 102 single mutants. On the other hand, if NER-mediated mutagenesis is through both GG-NER and 103 TCR, the combination of mutants lacking both Mfd and NER proteins would further reduce 104 mutation rates. To discern between these possibilities, we measured and compared the mutation 105 rates of the single and double mutants side-by-side. We found that the mutation rates of strains 106 lacking both Mfd and all three canonical NER factors have the same mutation rates as each 107 single mutant alone (Fig. 1B). This strongly suggests that all (mutagenic) NER is coupled to 108 transcription.

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To determine whether the mutagenic nature of NER is conserved across bacterial species, we 110 next determined the mutation rates of similar strains in Salmonella enterica serovar 111 Typhimurium, a Gram-negative bacterium which is highly divergent from B. subtilis. When we 112 compared the mutation rates of wild-type strains to those that either lack a Uvr protein or Mfd, 113 we found results that were consistent with what we observed in B. subtilis. In the absence of 114 UvrB, there was a similar decrease in mutation rates compared to strains lacking Mfd. These 115 results indicate that, the mutagenicity of NER is conserved amongst bacteria (Fig. 1C). 116 We have shown in the past that, over time, relatively small (in this case a 50-75% decrease) 117 differences in mutation rates can translate into large differences in the rate of evolution 2 . We 118 therefore used a laboratory evolution assay we previously established to determine whether the 119 mutagenic nature of NER accelerates evolution. For this, we measured adaptation to the 120 transcription inhibitor rifampicin in wild-type strains to those lacking UvrA or Mfd, or both, in 121 B. subtilis 2 . We have previously shown that the increase in the minimal inhibitory concentration 122 (MIC) observed over time correlates with the appearance of mutations in known resistance 123 genes 2 . After six days (approximately 40 generations), the median concentration of rifampicin 124 wild-type cells were able to survive was 3.2 µg/ml, while this was markedly lower for uvrA null 125 (0.4 µg/ml), mfd nulls (0.5 ug/ml), and uvrA mfd double mutants (0.3 µg/ml) (Fig. 1D). This 126 indicates that the differences observed in the mutation rates in NER deficient cells are 127 compounded and ultimately have large effects on adaptive evolution. In addition, these results 128 support the observation that all mutagenic NER is in the same pathway as Mfd, and again, most 129 likely universally transcription dependent.

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NER is generally thought to be an error free pathway. However, our observations clearly suggest 132 that this is not the case in the endogenous context. Our data suggest that the gap filling step of 133 NER is completed by an error-prone mechanism. During NER, gap-filling synthesis is the last 134 step of the pathway and based on in vitro experiments that examined DNA polymerase I (PolA in 135 B. subtilis), the dogmatic view became that this is the polymerase that functions during this step 5 . 136 Thus, we measured mutation rates in cells lacking PolA. Although in vitro work had led to the 137 conclusion that PolA is a high-fidelity polymerase 14 , we found that this is not the case in vivo. 138 We observed that PolA is mutagenic, as cells lacking PolA showed a decrease in mutation rates 139 that were very similar to that seen in NER deficient strains.

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To determine whether NER is mutagenic due to PolA activity, we measured the mutation rates of  determined the mutation rates of strains that either lacked PolY1 or PolY2, we didn't observe a 165 decrease in mutation rates in either single mutant. Interestingly we did observe a decrease in 166 mutation rates in strains lacking both PolY1 and PolY2, suggesting a redundant, pro-mutagenic 167 role for these polymerases (Fig 2B). If our hypothesis that these polymerases cooperate with 168 PolA during the NER gap filling step is correct, then in strains that lack all three polymerases, 169 we should not observe any additional decrease in mutation rates. Indeed, we observed that there  Fig 1B). Therefore, we conclude that these polymerases are in the same pathway and 173 cooperate to complete the last step during NER.

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The observed requirement for both an A-family replicative polymerase and a Y-family 175 polymerase led us to the hypothesis that PolA performs DNA synthesis during NER, but that it 176 often stalls at a DNA lesion that is present on the opposite strand to the original lesion that was 177 excised. This stalled PolA would then recruit a Y-family polymerase to overcome this lesion, 178 further increasing the chances of generating a mutation. More specifically, this DNA lesion 179 would be on the NER template strand, which is the non-transcribed strand. Therefore, it is 180 possible that the origin of this secondary damage stems from the non-transcribed strand being 181 single stranded during transcription and/or NER, which would make it more susceptible to DNA 182 lesions ( Fig 2C) 16,17 .

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To test this model, we again turned to our biochemical assay where we used purified PolA and 184 measured DNA synthesis on a template where we introduced an abasic site on the opposite 185 strand to that which is removed by the NER protein UvrD. We observed that an abasic site, one 186 the most common lesions observed in DNA 18 , is a strong block to synthesis by PolA 9 (Extended 187 data Fig 1C), supporting the model that PolA alone cannot fill in a gap generated during NER 188 that has damage on the non-transcribed strand. To complete gap-filling at this stage, Y-family 189 polymerases are most likely required, consistent with the results described above.

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Oxidative stress drives NER-dependent evolution 191 In this study, all mutation rates and evolution assays were performed in the absence of  To test our model, we performed evolution assays where we decreased the levels of oxidative 198 stress that the cells experience using two different methods. First, we performed evolution assays 199 in the presence of the antioxidant thiourea. This is an antioxidant molecule that has been used in 200 the past to reduce oxidative stress in bacteria 20-22 . We used concentrations that do not affect the 201 growth rate of our strains (Extended Data Fig 2A). We observed a marked decrease in the rate of 202 evolution of wild-type cells to various antibiotics when thiourea was present in the media (Fig.   203 3A, Extended Data Figure 2B). To confirm that the effect we observed was not simply due to 204 some artifact generated by thiourea, we also made a B. subtilis strain that overexpresses katA, the 205 gene that codes for the vegetative catalase, which breaks down hydrogen peroxide 23 . Similar to 206 the results we obtained with thiourea, we observed a decrease in the rate of evolution to the 207 antibiotic rifampicin (Fig. 3A). These results together strongly suggest that oxidative DNA 208 damage is indeed driving evolution. For both thiourea and katA overexpression, the decrease in the rate of evolution was similar to 216 those observed in strains lacking NER proteins. We therefore tested whether oxidative damage is 217 responsible for the mutagenic nature of NER. We performed evolution assays in strains deficient 218 in NER genes (uvrA nulls and mfd nulls). Although these strains have a serious deficiency in 219 evolving resistance to antibiotics, towards the end of the evolution assays, a slight increase in 220 their MIC can be observed (Fig. 3B, C). We took advantage of this and analyzed the rate at 221 which evolution starts to take off at the last time points when oxidative stress is reduced.
222 Consistent with our model, we observed that, in strains lacking Mfd, or the NER proteins, or 223 both, thiourea didn't have any effect on the rate of evolution. This strongly suggests that NER 224 mutagenesis and subsequent adaptive evolution depends on endogenous oxidative stress. 225 Last, to determine whether this was a conserved mechanism, we performed evolution assays in 226 patient derived strains of Pseudomonas aeruginosa (CF127) 24 and multidrug resistant 227 Staphylococcus aureus (MRSA). Again, we performed evolution assays, with and without 228 thiourea, at a concentration that again doesn't affect the growth rate of these bacterial species 229 (Extended Data Fig 2C, D). Consistent with our observations in B. subtilis, we found that in 230 these other species, there was a significant decrease in the rate of evolution when thiourea was 231 included in the media (Fig 4A, B). This effect is particularly striking in the case of the MRSA 232 strain, for which the concentration of antibiotic in which it could survive increased ≈250,000X 233 (Fig 4B) in the absence of thiourea. This increase was 32X for B. subtilis (Fig 3A) and ≈250X 234 for P. aeruginosa (Fig 4A). We have shown that nucleotide excision repair (NER), which strongly suppresses mutagenesis in 242 cells exposed to DNA damaging agents 3,4 , is actually promoting mutagenesis under endogenous 243 conditions and is generally a pro-mutagenic mechanism. Bacteria lacking any one of the three 244 core components of the NER mechanism, UvrABC, have lower mutation rates than wild-type 245 cells, indicating that NER causes spontaneous mutations (Fig. 1A, C). In addition, our data 246 indicate that all mutagenic NER functions in the same pathway as the transcription-coupled (TC) 247 NER factor Mfd, suggesting that NER is universally transcription dependent, at least under 248 endogenous conditions (Fig. 1B). This is consistent with recent biochemical findings regarding 249 NER and transcription and brings into question whether GG-NER is a mechanism that exists 10 .

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Interestingly, our data show that it is the cooperative nature of at least two DNA polymerases 251 that causes NER-dependent mutations: the replicative polymerase commonly associated with 252 NER, PolA, and one of two redundant Y-family polymerases, PolY1 and PolY2 ( Fig. 2A-C,

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Extended Data Fig 1C). We propose that DNA damage in the NER template strand (the non-254 coding strand, Fig 2C) explains this requirement for both DNA polymerases to complete NER, 255 which will naturally lead to an increased likelihood of mutations being introduced into the 256 synthesized DNA gap. This DNA lesion would be independent of the lesion that triggered NER, 257 and we propose that it is caused during transcription and NER, as the non-transcribed strand 258 stays as ssDNA for an extended period of time during both processes and it is well-known that 259 ssDNA is more prone to damage than dsDNA 16,17 .

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There is a potential alternative model: the NER machinery has been shown to excise non-261 damaged DNA in vitro 25 and that transcription stimulates this process in vivo 26 . These gratuitous 262 repair events, even if much less efficient than excision of damaged DNA, are predicted to be a 263 common phenomenon, as the amount of non-damaged DNA outweighs the amount of damaged 264 DNA by several orders of magnitude in cells that are not exposed to exogenous DNA damaging 265 agents 27 . Moreover, Mfd has been found bound to DNA throughout the genome in the absence of 266 exogenous DNA damage 28,29 , and it plays a role in transcription that is independent of its role in 267 TC-NER 30 . This constitutive association with DNA and RNAP could lead to excision, fill in 268 synthesis, and therefore increasing the likelihood of mutations being introduced onto undamaged 269 DNA.

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Interestingly, a recent pre-print has proposed that DNA lesions that are in close proximity and on 271 different strands lead to mutagenic NER, using a mouse liver cancer model in which cells are absence of thiourea, an antioxidant that has been shown to reduce oxidative stress in bacteria 20 . 287 We observed that the evolution of antibiotic resistance was much slower when thiourea was 288 present in the media for both B. subtilis and patient-derived pathogenic strains of P. aeruginosa 289 and MRSA (Fig 3A, Fig 4), indicating that oxidative stress drives evolution. Moreover, we 290 observed a similar effect when we overexpress the vegetative catalase katA (Fig. 3A), which 291 breaks down hydrogen peroxide, a molecule that can't itself react with DNA, but that can react 292 with free cellular iron which oxidizes DNA 38 . Interestingly we observed that the little evolution 293 we observe in NER deficient strains is not diminished in the presence of thiourea, supporting our 294 model that NER-dependent mutagenesis is mostly due to lesions caused by oxidative stress. This 295 is consistent with the mutagenic footprint of NER observed in mammalian cells 31 . Therefore, in 296 addition to showing that NER is universally mutagenic and transcription-associated in bacteria, 297 our findings likely explain the main source of NER-dependent mutagenesis not only in bacteria, 298 but also in higher eukaryotes.    Table 2).

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Determination of the mutation rates by fluctuation assays 438 Mutations rates were calculated as previously described 2 . A single colony was inoculated into 2 439 ml of LB and grown for 2 hours (B. subtilis) or 2.5 hour (S. enterica) to reach exponential