Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli

Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators.

Introduction Spontaneous mutations drive development of most cancers and their resistance to therapy, aging, pathogen escape from the immune response and antibiotics, and evolution generally. Although central in all of biology and many aspects of human health, the causes of spontaneous mutagenesis have long been elusive, and generally difficult to assign with confidence. Whereas many ways to increase or induce mutagenesis are known, the origins of spontaneous mutations in most organisms remain speculative [1,2].
Spontaneous mutations could potentially form by many different mechanisms [1,2]. An early proposal [3] was that most spontaneous mutations result from spontaneous DNA damage-that repair of damaged DNA is more mutagenic than standard DNA synthesis. The mutagenicity of DNA repair has been borne out, for example, by demonstrations that repair of DNA doublestrand breaks (DSBs) is mutagenic in bacteria [4][5][6][7][8], yeast [9], and human cancer [10][11][12] (reviewed [13][14][15][16]). That DNA damage underlies much of spontaneous mutagenesis was supported by discoveries that yeast antimutator mutants, i.e., mutants with lower-than-normal spontaneous mutation rate, carry mutations in DNA-damage-survival genes [17]. These genes encode alternative error-prone DNA polymerases or proteins that assist them, allowing survival of DNA damage by replication over or extension from otherwise-replication-inhibiting damaged DNA bases [18], implying that most spontaneous mutagenesis in yeast results from use of errorprone DNA polymerases during DNA-damage survival. To our knowledge, the only mechanistic detail available on spontaneous mutagenesis mechanisms is that about half of all spontaneous base-substitutions and small insertion/deletions (indels) in starving Escherichia coli form dependently on the proteins used in stress-inducible mutagenic DNA break repair (MBR) [7].
In MBR, alternative error-prone DNA polymerases appear to be switched into the DSBrepair replisome under the control of stress responses [6][7][8] (reviewed [13,15,16]). The central features of this mechanism have been recapitulated biochemically with purified proteins [19]. Repair of DSBs by homologous recombination requires high-fidelity replicative DNA polymerase III in unstressed cells [20]. In MBR, DSB repair uses alternative error-prone DNA polymerases, principally Pol IV (DinB) [21], but also Pols II [22] and V [7,23], under the control of the SOS DNA-damage response and the general/starvation stress response controlled by the RpoS transcriptional activator. The sequence signature of RpoS-dependent MBR is evident across bacterial genomes [24], implicating this mechanism or similar mechanisms in most bacterial evolution. Moreover many stress-induced mutation mechanisms from bacteria to human cancer cells show similarities to E. coli MBR [13]. From these previous studies, it seemed probable that the critical step leading to MBR, and perhaps spontaneous mutagenesis generally, could be the switch from high-fidelity replicative DNA polymerases to error-prone DNA polymerases, resulting from DNA-polymerase competition promoted by upregulation of those DNA polymerases, in E. coli, by the SOS and general stress responses [13,15,16]. Here, we show that, for MBR, it is not sufficient to upregulate mutagenic DNA polymerases and repair DSBs; damaged DNA bases are also required.
The presence of 8-oxo-dG in DNA results both from incorporation of oxidized dGTP from the deoxynucleotide triphosphate (dNTP) pool and from in situ oxidation of guanine (G) in DNA [34]. 8-oxo-dG is often incorporated opposite to, and templates incorporation of, an adenine (A) leading to A to C and G to T transversion mutations (e.g., [35,36]). If 8-oxo-dG is incorporated into DNA, it can persist or be excised by base excision repair (BER). Although replication bypass of template 8-oxo-dG has been shown to occur with high efficiency [35,37], it is found that the eukaryotic replicative polymerase pol δ is transiently inhibited at 8-oxo-G [38]. Pol δ is able to extend from the A:8-oxo-dG base pair, but not from C:8-oxo-dG. A switch to an alternative polymerase, often pol λ, allows extension from C:8-oxo-dG with no substitution mutation [38]. In E. coli, of the alternative DNA polymerases, Pol IV is responsible for most MBR mutations [21], and makes base substitutions and indel mutations, mostly 1 basepair deletions in mononucleotide repeats [39].
Pol IV is reported to make mutations in the absence of induced DNA damage when overproduced [39,40], and SOS-upregulated levels of Pol IV are required for MBR [21,41]. Here, we report that SOS-and general-stress-response induced overproduction of Pol IV is not sufficient for Pol IV-dependent MBR. Damaged bases in the DNA must also be present. We hypothesize that damaged bases allow the switch to use of Pol IV during MBR by pausing the replicative polymerase to allow polymerase exchange in the replisome. The findings suggest that spontaneous mutation by MBR is regulated by regulation of the intracellular level of ROS.

MBR assay in E. coli
The E. coli Lac assay [42] for MBR quantifies both indels and gross chromosomal rearrangements (GCRs) as reversions of a lacI-Z+1bp frameshift allele in an F' conjugative plasmid.

Reduction of ROS through exogenous agents reduces MBR
We reduced ROS levels in cells undergoing MBR using 2,2'-bipyridine (bipyridine) and thiourea (TU), chemical agents commonly used to reduce ROS levels in living cells. Both agents enter cells and prevent ROS formation (bipyridine) or quench ROS (TU) [54]. Bipyridine chelates ferrous iron and prevents it from catalyzing ROS-forming Fenton reactions [55]. TU quenches hydroxyl radicals after formation, and prevents them from damaging macromolecules [56]. We added varying amounts of bipyridine (0.1 uM and 0.2 μM) or TU (25, 50 and 100 mM) to solid minimal lactose medium on which MBR occurs, and found that Lac + mutation rates were reduced dose-dependently by either ROS reducer, with a 90% ± 4.3% reduction at 100 mM TU (Fig 1A and 1B) (p = 5.2x10 -5 , Student's 2-tailed t-test). TU also prevented the previously reported increase in MBR in cells lacking Dps [57], a stationary-phase nucleoidcompaction protein that protects DNA against ROS, again in a dose-dependent manner ( Fig  1C). There is a 40% ± 7.8% reduction caused by 0.2 μM bipyridine (Fig 1D and 1E) (p = 0.02, Student's 2-tailed t-test). Neither bipyridine nor TU affected viability of Lacmutationreporter cells over the duration of incubation, nor did they affect the time required for formation of Lac + colonies under precise reconstructions of experimental conditions (S1 Fig), indicating that mutation rate, not ability of treated cells to form colonies, was reduced by lowering ROS levels. All reductions in mutagenesis affected SNAs and amplification proportionately (an example is shown in Fig 2A). These data imply that ROS are required for MBR and suggest that Dps might inhibit mutagenesis by preventing ROS damage to DNA [57].

Production of ROS-detoxifying enzymes reduces MBR
We reduced ROS levels in cells undergoing MBR by constitutive overexpression of either of two E. coli oxidative stress responses: the SoxRS response and the OxyRS response [31, 32]. Whereas each regulon responds to the presence of ROS in the cell, they employ different enzymes that detoxify different radical species. The SoxR response upregulates superoxide dismutases that inactivate superoxides and their derivatives [31], and the OxyR response upregulates catalases and alkylhydroperoxidases that inactivate hydrogen peroxide and its radical derivatives including hydroxyl radicals [32]. We used the oxyR2 [58] and soxR104 [59] alleles (separately), which cause constitutive expression of each response at induced levels, in the absence of oxidative inducers, to reduce ROS levels normally present during MBR. soxR104 reduced Lac + MBR mutation rate, conferring a 90 ± 5.5% reduction (Fig 2B and 2C) (p = 0.004, Student's 2-tailed t-test). Depression of MBR by oxyR2 (59 ± 11%) was not quite significant at the 5% level (p = 0.07, Student's 2-tailed t-test) when tested in the wild-type strain. However, oxyR2 completely eliminated the Δdps-mediated increase in Lac + MBR, showing a 70 ± 7.8% reduction in mutation frequency (Fig 2B and 2D), (p = 0.014, Student's 2-tailed ttest) to the same level as the strain with oxyR2 alone (p = 0.98, Student's 2-tailed t-test). The data indicate that ROS are required for MBR generally, and we infer that the mechanism of Dps inhibition of MBR is its protection of DNA against ROS.

ROS are required for stress-induced mutagenesis
We removed ROS from cells by overexpressing sodB, which encodes a superoxide dismutase. We used a plasmid from the mobile plasmid library [60] that carries each gene under the , depicted here normalized to the wild-type, are reduced proportionately by soxR104 and oxyR2 alleles, which constitutively activate the SoxR and OxyR responses respectively. Indels (SNAs) and GCR were distinguished (Methods) in all experiments, and no disproportionate effects were detected. (B) Representative experiment. (C) Quantification of MBR rates from three experiments: soxR104 reduced MBR by 90% ± 5.5% (p = 0.004, Student's 2-tailed t-test). oxyR2 reduced MBR but not quite significantly at the 5% level when tested in the wild-type (59 ± 11%), p = 0.07, Student's 2-tailed t-test). (D) oxyR2 reverses the hypermutation phenotype of Δdps cells. * indicates significantly different from wild-type (p 0.05). Wild-type, SMR4562, black; soxR104, PJH2947, red; oxyR2, PJH3278, blue; Δdps, PJH2608, pale green; oxyR2 Δdps, PJH3295, dark green. The data indicate that ROS are required for mutation formation, and that the hypermutation seen in Δdps cells depends on decreased protection from ROS. control of an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter in cells used for the Tet MBR assay [7]. The Tet MBR assay reports on indel mutations that revert a chromosomal tetA +1bp frameshift allele during starvation in liquid minimal medium. In the Tet assay, DSBs are induced near a chromosomal tet mutation-reporter gene by the I-SceI site-specific double-strand endonuclease expressed in F-plasmid-free cells during starvation. tet reversion in this assay requires the key MBR proteins, as in Lac + MBR, including RpoS, the SOS response regulators, Pol IV, and DSB-repair proteins [6,7,16]. The Tet assay differs from the Lac MBR assay not only in having no F-plasmids present, but also in having no selection for the reverted allele during the starvation stress; Tet-resistant (Tet R ) revertants are selected as Tet R cfu after the cells are rescued from starvation in liquid. The Tet assay does not measure GCR. We found that over-expression of sodB reduced Tet R MBR mutant frequencies 76% ± 12%, (Fig 3, 0.003, Student's 2-tailed t-test). These data show, by enzymatic removal of ROS, that ROS are required for starvation-stress-induced MBR. We conclude that MBR requires ROS for mutagenesis.

ROS promote MBR other than by formation of DSBs
We eliminated several possible mechanisms for the requirement for ROS in MBR. Repair of oxidized DNA bases might lead to formation of spontaneous DSBs that instigate MBR [4,6,7,47]. Base excision repair proceeds by excision of the damaged base by specific DNA In the Tet MBR assay, DSBs are induced in starved cells by induction of I-SceI double-strand endonuclease cleavage at an I-SceI cutsite near the chromosomal tet indel mutation-reporter gene [6,7]. SodB overexpression is induced by IPTG, and reduces TetR mutant frequency even when DSBs are provided by I-SceI. Thus, I-SceI cannot substitute for ROS, and ROS promote MBR other than or in addition to by causing DSBs. * significantly different from the isogenic emptyvector control (pVector) at the 5% level. ΔrpoS and dinB50 positive-control strains with empty vector and DSB induction demonstrate RpoS-and DinB-dependent MBR, here and in glycosylases forming an abasic (AP) site, followed by nicking at that site by an AP endonuclease. If not repaired, the nick could produce a one-ended DSB when replicated, via replication fork breakage [61]. Using the Tet MBR assay, we find that even in the presence of an I-SceIendonuclease-generated DSB, over-expression of sodB still reduced Tet R MBR as described above (Fig 3). We show that induction of DSBs by I-SceI is effective when SodB is overexpressed (S2 Fig). We conclude that ROS promote MBR other than or in addition to by promoting spontaneous DSBs.

ROS do not promote MBR via oxidation of proteins
ROS damage proteins, lipids, RNA and DNA [30]. Oxidation of proteins, which causes carbonyl groups, can inhibit protein function (reviewed by [33]). We used a mutation that reduces cellular levels of carbonylated proteins: rpsL141, which encodes a hyper-accurate ribosomal protein that reduces intracellular carbonylated protein content [62]. We found that Lac assay MBR rates were unaffected by rpsL141 (Fig 4A and 4B), implying that promotion of MBR by ROS is not mediated by oxidation of proteins.

ROS promote MBR other than via induction of the general or SOS stress responses
We excluded the possibility that ROS promote MBR by inducing the RpoS response by assaying MBR in an rssB mutant in the presence of TU. RssB targets RpoS, the transcriptional activator of the general stress response, for degradation by the ClpXP protease, so that when rssB is deleted, RpoS levels increase, artificially upregulating the RpoS response [64]. If ROS promoted MBR solely by inducing the general stress response, ΔrssB would be expected to counter the anti-MBR effect of TU treatment. We find that TU treatment reduced MBR dose-dependently in ΔrssB cells as in wild-type cells (Fig 4C), (p = 0.16; 0.92; 0.95, comparing WT with rssB at 25, 50, and 100 mM TU, respectively; Student's 2-tailed t-test). We conclude that ROS probably promote MBR other than or in addition to by activation of the general stress response.
The SOS DNA-damage response promotes MBR via its 10-fold transcriptional upregulation of Pol IV error-prone DNA polymerase, and is not needed for MBR in cells with an operatorconstitutive (constitutively over-expressing) dinB allele [41]. We find that the dinB(o c ) allele did not counter TU treatment [ Fig

Oxidative damage promotes MBR other than by titration of mismatch repair
We tested the possibility that excessive base damage from ROS might overwhelm mismatch repair, making it unable to correct base misincorporations by DinB. MutL, a required component of mismatch repair, was shown to become limiting during MBR [63] and for polymerase errors generated by a hyper-mutator Pol III [65]. We combined TU treatments with overproduction of MutL, to test whether oxidized DNA bases saturate mismatch repair capacity during MBR. Previously, increasing mismatch repair capacity by overproducing MutL reduced Lac + MBR approximately 4-fold [63]. If ROS promoted MBR by saturating mismatch repair capacity, MutL overproduction and TU inhibition of MBR would be expected have an epistatic relationship, implying their action in the same pathway. By contrast, we found that 100mM TU combined with MutL overproduction reduced Lac + MBR additively ( Fig 4E), P = 0.005, 0.002, 0.838, and 0.035 for WT compared with MutL overproduction; WT compared with TU; WT + TU compared with MutL overproduction; and WT + TU compared with MutL overproduction + TU, respectively, Student's 2-tailed t-test. The data imply that ROS promote MBR other than by causing saturation of mismatch repair.

ROS promote MBR via persistent oxidized guanine in DNA
We found that removing oxidized guanine from DNA reduces MBR. E. coli has three proteins that specifically reduce the mutagenic effects of 8-oxo-dG. MutM is a DNA glycosylase that excises 8-oxo-dG from DNA, MutY is another glycosylase that excises mispaired adenine opposite to G or 8-oxo-dG, and MutT hydrolyses 8-oxo-dGTP in the nucleotide triphosphate pool, reducing incorporation of 8-oxo-dG into DNA. We (separately) overproduced MutM, MutT, and MutY in Tet MBR assay cells using mobile plasmid library plasmids [60], and measured Tet R mutant frequencies. We detected no decrease in cell viability during these experiments or in growth of strains containing these plasmids induced with 1mM IPTG (S3 Fig). We found that overproduction of MutM and MutT reduced Tet R MBR 82% ± 9.7% and 91% ± 5.3%, respectively, compared with the vector-only control (p = 0.0015 and 0.0002, Student's 2-tailed t-test) (Fig 5). ΔrpoS and dinB positive-control strains confirm that mutagenesis is via Overproduction of MutM and MutT reduced Tet R mutant frequency 82% ± 9.7% and 91% ± 5.3%, respectively compared with the vector control (p = 0.0015 and 0.0002, Student's 2-tailed t-test). The data indicate, first, that 8-oxo-dG in DNA is required for MBR, second, that the effects of 8-oxo-dG on MBR result mainly from incorporation of 8-oxo-dG from the dNTP pool, and third, that 8-oxo-dG must remain in DNA for MBR to occur, because if 8-oxo-dG is removed by the MutM DNA glycosylase, MBR is reduced. * significantly different from the empty vector control (pVector) at the 5% level. DNA base damage and mutagenic break repair the canonical MBR pathway. In eight repeated experiments we observed a small, but not significant, decrease in Tet MBR with MutY overproduction (32% ± 9.5% decrease, p = 0.15, Student's 2-tailed t-test), suggesting that mispaired adenines have, at most, a minor effect on promotion of MBR. We conclude that persistent unrepaired 8-oxo-dG in DNA is required for MBR, and that ROS promote MBR via the presence of 8-oxo-dG in DNA. Furthermore, because the increased removal of 8-oxo-dG from DNA eliminates most mutagenesis, we conclude that whereas oxidized proteins or lipids, or oxidative lesions in RNA might play a role in MBR, it is at most a small one. The strong effect of overproduction of MutT, the 8-oxo-dGTP diphosphatase, implies that the MBR-promoting 8-oxo-dG in DNA results mainly from incorporation from the nucleotide pool rather than in situ oxidation of DNA.
Other base damage in DNA can substitute for 8-oxo-dG in MBR We tested the hypothesis the 8-oxo-dG in DNA might promote MBR by pausing the highfidelity replicative DNA Pol III, active in DSB repair [20], which might allow Pol IV and other alternative DNA polymerases to switch into the active position in the repair replisome. If this were the case, then 8-oxo-dG would not be required as a specific intermediate in MBR; any fork-stalling DNA-base damage traversed or extended from by translesion DNA polymerases would be expected to substitute for 8-oxo-dG. We tested whether other persistent DNA damage could bypass the requirement for ROS and 8-oxo-dG in MBR by using methyl methanesulfonate (MMS), which methylates DNA, forming mainly N 7 -methyldeoxyguanosine and N 3methyldeoxyadenosine [66], and ultraviolet-C light (UV-C), which creates pyrimidine dimers and 6-4 photoproducts (reviewed by [67]), all of which stall the replicative DNA Pol III and can be traversed by translesion DNA polymerases [68][69][70]. We pulse-treated starved cells with nonlethal doses of MMS or UV-C before plating on minimal lactose medium with 100mM TU to scavenge ROS. Whereas both MMS and UV-C increased basal mutation rates slightly (3-fold for each treatment, Fig 6A and 6B), the increase in mutagenesis seen with TU + MMS or UV treatments compared with TU alone was many-fold greater (Fig 6C), 22 ± 7.8-fold for 10mM MMS (p = 0.02, Student's 2-tailed t-test), and the increase is 16 ± 2.3-fold for 10 J/m 2 UV, (p = 0.001, Student's 2-tailed t-test). The data show a robust return to mutagenesis caused by those base-damaging agents after removal of ROS. The data imply that other base damages can substitute for 8-oxo-dG in mutagenesis. MMS or UV treatment completely countered the effect of 100 mM TU ( Fig 6B). Further, we show that the mutagenesis restored by MMS or UV in the absence of ROS is "on-pathway" MBR, in that mutagenesis depends completely on RecA, RpoS, Pol IV, and RuvC ( Fig 6D). These data show that MMS and UV did not activate an alternative mutagenesis mechanism(s), but rather restored MBR to ROS-depleted TUtreated cells.
Both MMS and UV-C might induce oxidative damage in addition to their more common lesions. UV-C can induce 8-oxo-dG formation in HeLa cells and in DNA, although 8-oxo-dG induction was about 2000-fold lower than pyrimidine dimer induction [71]. MMS induces ROS in yeast [72], however, this requires the presence of glucose in the medium [73,74], and glucose is absent from the medium of starved E. coli cells, making this unlikely. In Fig 1D we used a high dose (100mM) of TU to scavenge ROS and found that the four-fold increase in MBR caused by Δdps is completely blocked by 100mM TU. Given the expected much greater levels of ROS in Δdps than WT cells and the ability of TU to quench the ROS in Δdps cells, these data suggest that there is still substantial ROS quenching capacity when TU is applied to wild-type cells. All of these data imply that MMS and UV-C promoted MBR not by promoting 8-oxo-dG in DNA, but rather by their abundant standard base damages. The slightly lower mutagenesis seen in the presence of TU for all mutagen treatments in Fig 6B (p = 0.01, 0.03, 0.40, and 0.20, Student's 2 tailed t-test for 5 J/m 2 UV, 10 J/m 2 UV, 5 mM MMS, and 10 mM MMS, respectively, in the presence versus the absence of TU) might reflect a low level of MBR from oxidative damage when UV and MMS are used. However, most MBR in MMS or UV treated cells arises from non-oxidative base damage, which is not prevented by TU. These data demonstrate that persistent damaged bases in DNA are needed generally for MBR, and imply that the DNA substrate for MBR is more than a simple DSB undergoing HR repair: that, additionally, damaged DNA bases must be present in the DNA for MBR to occur. , there is an 11 ± 0.6-fold increase (p = 0.0004, Student's 2-tailed t-test), compared with TU treatment with no mutagen. For 10 J/m 2 UV, the increase is 16 ± 2.3-fold (p = 0.001, Student's 2-tailed t-test; for 5mM MMS, 28 ± 6.6-fold (p = 0.049, Student's 2-tailed t-test) and for 10mM MMS, 22 ± 7.8-fold (p = 0.02, Student's 2-tailed t-test). (C) MMS and UV treatments have much stronger effects on Lac + mutant formation when ROS are scavenged by TU than when they are not. Although both treatments increased basal Lac + mutation rates, 3 ± 0.5-fold with 10 mM MMS and 3.0 ± 0.8-fold with 10 J/m 2 UV, the increases caused by MMS or UV were much greater in TU treated than untreated cells, indicating that MMS and UV treatments specifically substitute for the requirement for ROS (p = 0.006 for both, Student's 2-tailed t-tests, when comparing mutagen +TU/ NT+TU with mutagen only/NT with no mutagen). Data are from Fig 6B. (D) The mutagenesis restored to ROS-depleted cells by UV and MMS is on-pathway MBR, requiring RpoS, RecA, DinB and RuvC. Thus, the requirement for damaged bases in DNA for MBR is not specific to 8-oxo-dG, but can be provided by other replication-hindering DNA damage. Strains used: Wild-type (WT), SMR4562; ΔrpoS, PJH1399; recA, SMR4610; dinB50, SMR5889; ruvC, SMR6906. https://doi.org/10.1371/journal.pgen.1006733.g006

Discussion
Discovered in E. coli, MBR is an important model molecular mechanism of mutagenesis later discovered in other bacteria, yeasts, flies, and human cells and cancers (reviewed [13]). Until this report, the E. coli MBR mechanism was defined by its requirements for various trans-acting components, but only a single central cis-acting DNA component: DNA DSBs, which are required for spontaneous MBR [4,6,7,47] and at which mutagenesis is focused [6,8]. The trans-acting proteins are those of DSB repair, low-fidelity translesion DNA polymerases, transiently insufficient mismatch repair and stress-response regulators (reviewed [13]), including a large gene network, in which most act upstream of the stress-response regulators in stress sensing and signal transduction that activates the RpoS, SOS and membrane stress responses [75]. Supporting this binary view, although transcriptional R-loop RNA/DNA hybrids and the Mfd RNA polymerase translocase are also required for MBR [47], they and the membrane stress response [45] promote MBR by promoting spontaneous DSBs at some loci, and are not needed when endonuclease-generated DSBs are provided [47,52]. By contrast, the data presented here show that a separate, additional and unrelated DNA substrate and event must occur for MBR: damaged DNA bases. We found that spontaneously, during starvation-induced MBR, ROSpromoted 8-oxo-dG in DNA underlies most MBR (Figs 1-3 and 5), and that 8-oxo-dG does not promote MBR solely by generation of DSBs (Figs 3 and 5), and so is a distinct, new cis-acting DNA component of the MBR molecular mechanism. This mechanism applies both to SNA and GCR MBR mechanisms (Fig 2A). Other roles for ROS in stress response activation, protein oxidation, and mismatch repair saturation were shown not to underlie the ROS role in MBR (Fig 4). Because the persistent 8-oxo-dG in DNA that drives spontaneous MBR during starvation can be substituted by other generic damaged bases caused by UV light or the alkylating agent MMS (Fig 6), the data indicate that damaged bases generally are required for MBR, and in a role separable from specifically oxidized guanine.
Whereas previously we found that the stationary-phase upregulated ROS-detoxifying protein Dps inhibits MBR [57], here we find that the Dps inhibition of MBR is consistent with its effects on ROS reduction (Figs 1C and 2D).

Model: Damaged bases promote DNA polymerase switching to license MBR-SNAs
We suggest a model in which the role of damaged bases in MBR is to allow the switch from high-fidelity DNA Pol III [20] to low-fidelity translesion DNA polymerases during homologous recombinational DSB repair, thus allowing MBR. A requirement for Pol III pausing to allow a switch to Pol IV has been shown previously [76][77][78]. A simple example of this idea is illustrated in Fig 7 for the formation of SNAs. The general model is that replication stalling at base damages that are not easily replicated or extended from by Pol III allows switching to alternative DNA polymerases, some of which are mutagenic, by pausing the replisome so that the DNA polymerases can switch.
Damaged bases can be repaired in double-stranded DNA, but in single-stranded DNA at the replication fork they must be bypassed by translesion synthesis (TLS) [79]. TLS often uses alternative DNA polymerases to synthesize across damaged bases or to extend from the resultant mismatches after incorporation [80]. TLS polymerases include the Y-family polymerases Pol IV/DinB and Pol V in E. coli. DNA polymerases in the replisome and in repair complexes are attached to the β processivity clamp, which is the structural homolog of proliferating cell nuclear antigen (PCNA) in yeast and mammalian cells [81]. In both E. coli and eukaryotes, replicative polymerases might pause when they encounter a damaged base, either before incorporation or before extension, allowing a TLS polymerase that is already loaded on the β-clamp  [20] to any of the alternative mutagenic DNA polymerases results from Pol III pausing at a damaged base, either in the template or newly added to the 3'-primer end. One way that 8-oxo-dG could allow Pol IV-mediated MBR mutations (at other DNA bases) is shown here. (A) A D-loop made during DSB-repair of a one-ended DSB has invaded a sister DNA molecule (parallel lines, base-paired DNA) and primed new synthesis (red line) using Pol III. Pol III inserts 8-oxo-dG, here opposite a template C [38], or could stop after inserting C at 8-oxo-dG in the template (not illustrated). (B) Pol III pauses because it extends poorly from an 8-oxo-dG primer end paired with template C [38]. We suggest that the pausing allows Pol III to leave the active position on the β-clamp, and an alternative DNA polymerase on the β-clamp (Pol IV/DinB shown here) to move into the β-clamp active position. (C) Pol IV in the active position extends synthesis from the 8-oxo-dG primer opposite template C (or, not illustrated, bypasses an 8-oxo-dG in the template strand, inserting the correct base or an incorrect base), and continues synthesis making indel (and/or base substitution) errors at mononucleotide repeat sequences, at which it is most error-prone. (D) Eventually Pol IV pauses because of its limited processivity [39], allowing Pol III to resume. Black lines represent pre-existing nucleotide chains and red lines represent new synthesis. The β-clamp is represented as an orange doughnut. The β-clamp site with the active DNA polymerase is illustrated at the top of the β-clamp, and the [77] to move onto the DNA primer end and synthesize past the damaged base or to extend beyond it. In the version of this general model shown in Fig 7, we show Pol IV (DinB) replacing Pol III in the active position on the beta clamp (Fig 7B). The replicative Pol III can incorporate 8-oxo-dG opposite A (not illustrated) or C (Fig 7A) [38]. Assuming that findings about eukaryotic polymerases [38] can be applied to E. coli, Pol III would then pause transiently because of poor ability to extend synthesis from the 8-oxo-dG paired with template C [38]. Alternatively Pol III could pause at an 8-oxo-dG in the template strand paired with C on the primer (not illustrated) [38]. In these cases, we suggest that stalling of Pol III allows it to be shifted out of the β processivity clamp site carrying the active DNA polymerase (top of βclamp doughnut shown Fig 7), and a DNA polymerase in the clamp inactive position (bottom of β-clamp, Fig 7) switched into the active position (Pol IV shown switching places with Pol III, Fig 7B and 7C). With Pol IV in the β-clamp active DNA polymerase position, synthesis can resume from the 8-oxo-dG primer opposite template C (red line, Fig 7C) or from C across from 8-oxo-dG in the template (not illustrated), and base-substitution and/or indel mutations can be made downstream after the switch, for example when Pol IV encounters a run of Gs (Fig 7D and 7E), a slippery template sequence at which Pol IV makes errors with high probability [72]. Pol IV is processive for about 400 nucleotides [39]. Pol IV and its orthologous Yfamily DNA polymerases in other species including human can make indel mutations because their active sites can accommodate extrahelical bases in mononucleotide repeats in the template strand, and thus insert fewer nucleotides than are on the template [82,83]. Their most common errors are base substitutions, however. The data presented here reveal that the availability of alternative DNA polymerases is not sufficient to allow mutagenesis, which requires polymerase switch, supporting models like this one.

Alternative DNA polymerases in MBR
All three DNA damage-response (SOS)-induced polymerases, Pols II, IV and V, contribute to stress-induced MBR SNA formation [7,[21][22][23]. Pol IV promotes 85% of indels in Lac MBR, and can increase mutation rates over a thousand-fold in the absence of exogenously induced DNA damage when overproduced in E. coli [40]. The remaining 15% of Lac MBR indels require Pol II [22]. Pol V promotes MBR base substitutions [23] and some indels [7]. GCR, seen as amplifications, does not require any of the SOS-inducible DNA polymerases, but requires DNA Pol I [45]. Thus all stress-induced MBR requires alternative DNA polymerases. Presumably the relative availability of the different DNA polymerases will influence which polymerases are switched into the MBR replisome and replace Pol III at the active position on the beta clamp.

Model: Damaged bases promote DNA polymerase switching to license MBR-GCRs
Amplification in the Lac assay is mediated by microhomology [45] and is proposed to arise by polymerase template switching during replication by a microhomology-mediated breakinduced replication (MMBIR) mechanism [46,84]. Because Pol I is required for amplification [53], we suggest that Pol I moves to the beta clamp active position when Pol III is stalled ( Fig  8B and 8C), and then mediates template switching creating genome rearrangements. We suggest that when Pol I binds the primer end and the replisome is dispersed (Fig 8B, 8C and 8D),  as will happen if the fork is stalled for a time [85], Pol I can mediate template switching using very limited homology (Fig 8D and 8E), as has been observed for the Pol I human ortholog DNA PolQ, another A-family polymerase [86][87][88]. This mechanism (one specific example of which is illustrated in Fig 8), parallels the model for base-substitution and indel formation (one indel version shown in Fig 7). Dissociation of the primer end with Pol I attached might be achieved if the editing function of Pol I is activated by the mismatch at ROS damaged bases, for example at an 8-oxo-dG:C base pair, causing dissociation of the 3'-end to enable it to attain the nuclease domain [89]. Such dissociation would permit polymerase template switching (Fig  8E), the postulated first step in microhomology-mediated recombination.

Other possible ROS-induced mutagenesis mechanisms
Other possible mechanisms that might have explained the role of ROS in MBR have been described, but do not fit the data presented here. First, because oxidation induces nicks and DSBs directly in DNA [90,91], and both UV and MMS cause DNA breakage [92,93], together with the established requirement for DSBs for MBR [6], we considered the possibility that ROS were needed for MBR for the provision of DSBs. We answered this by showing that expression of I-SceI double-strand endonuclease did not raise MBR in a strain over-expressing the SodB superoxide dismutase to the level seen in a strain also expressing I-SceI but not overexpressing SodB (Fig 3). This shows that the loss of MBR associated with reduction in endogenous ROS is not suppressed by provision of DSBs, and therefore that ROS has a role in MBR other than or in addition to DSB formation. Similarly, the action of I-SceI did not suppress the reduction in MBR associated with over-expression of MutT (Fig 5), showing that removal of 8-oxo-dGTP from the deoxynucleotide triphosphate pool is not overcome by provision of DSBs. If ROS were required solely for DSB formation we would have seen MBR at wild-type levels in MutT over-expressing strains.
Second, Pol IV overproduction causes cell death by increasing incorporation of 8-oxo-dG into DNA with subsequent increase in DSBs [54]. This is postulated to be because Pol IV is more likely than replicative polymerases to incorporate 8-oxo-dG, which is then removed by base-excision repair in 8-oxo-dG clusters leading to DSBs [54]. This model cannot account for the role of ROS/8-oxo-dG in MBR because the requirement for 8-oxo-dG was other than or in addition to for generation of DSBs (Figs 3 and 5). Also arguing against the Pol IV/8-oxo-dG-DSB model is the observation that amplification also requires ROS (Fig 2A), and neither SOS-induced levels of Pol IV nor Pol IV itself are required for amplification [21]. Thus the model of Foti et al. [54] does not provide an explanation for the requirement for ROS for MBR. Third, a model for how single-stranded (ss) DNA damage promotes MBR in yeast also does not explain our data in E. coli. In yeast, ssDNA regions experience hypermutability both spontaneously and induced by UV light or MMS treatment [94], with mutagenesis increased by orders of magnitude in ssDNA regions at sites of DSB repair by HR, compared with duplex DNA nearby. The ssDNA at sites of HR DSB repair and at uncapped telomeres involved ssDNA lengths longer than 6kb [95]. A similar mechanism is implicated in human cancer cells [12]. This hypermutability in ssDNA is attributed to the absence of complementary DNA sequence during repair of base lesions formed in the ssDNA, causing their replication by mutagenic translesion DNA polymerases [95]. This model appears not to apply to MBR in E. coli both because the E. coli MBR mutation clusters cover~100 kb rather than six [8], and because the model offers no explanation for GCR in MBR.
Finally, ROS were reported previously to participate in an RpoS-independent mechanism of starvation-associated mutation in E. coli [96]. In contrast with our findings for RpoS-dependent MBR, in that assay, although mutating mutT, encoding the 8-oxo-G nucleotide pool sanitizer, increased mutagenesis, mutation of mutM, encoding the 8-oxo-dG DNA glycosylase, showed no significant effect, showing no role for 8-oxo-dG in DNA [96,97], the opposite of our finding (Fig 5). Thus, a different mutagenesis mechanism was at work.

Roles of ROS and damaged bases in spontaneous mutagenesis
Importantly, the mutagenesis-promoting effect of ROS and 8-oxo-dG in DNA reported here is in spontaneous mutation. Although increasing ROS in cells induces mutagenesis (e. g. [25-27]), whether basal levels of spontaneous mutation are also driven by ROS was unknown previously. Previously, the sequence spectrum and frequencies of mutations in anaerobic and aerobic E. coli cultures suggested that spontaneous base substitutions were oxygen-related, via hydroxyl radicals [98]. The data here show directly that spontaneous SNA and GCR MBR in starving cells require ROS and 8-oxo-dG.
A large body of work has established that organisms respond to stress by increasing their mutation rate under the control of stress responses [13] possibly increasing their ability to evolve and adapt [99]. The demonstration that mutation rate in stressed cells additionally requires base damage caused by ROS, together with our previous finding [57] that there is both positive (by H-NS) and negative (by Dps) regulation of ROS, suggest that cells might regulate their spontaneous mutation rates in response to challenging circumstances by regulating/responding to ROS levels. ROS were long regarded as unwanted byproducts of oxidative metabolism, to be avoided because of the damage that they do to macromolecules. They have been held to be a primary cause of aging [100,101]. However, it is now apparent that ROS are also an integral component of cell physiology [102]. The use of ROS for accelerated evolution in stressed cells described here might be regarded as a parallel with the involvement of ROS in signaling [103] and in the function of the immune system [104]. Conversely, although it might be advantageous for an organism to mutate specifically when under stress [99,104,105], it would be to our advantage to stop pathogens from responding to our immune systems and antibiotics with hypermutation, evasion of the immune system and antibiotic resistance. We have suggested that novel "anti-evolvability" drugs might stop stress-induced evolution of pathogens (and cancers) by suppression of the stress-responses that promote mutagenesis [13].

Construction of Esherichia coli K12 strains
The origin of these strains is listed in Table 1. Strains used for Lac + MBR assays are isogenic derivatives of SMR4562, an independent construction of FC40 [106]. Genotypes were confirmed either by PCR or by sequencing as necessary. Mobile plasmid library plasmids [60] containing over-expression alleles of mutants under the control of a P tac promoter were conjugated into SMR10866 and SMR10798, strains expressing the chromosomal I-SceI restriction endonuclease controlled by P BAD , and an I-SceI cutsite, or the enzyme-only control, respectively. Others were constructed by transduction or linear replacement [107] as listed in Table 1. I-SceI cutting was verified by the sensitivity of strains to arabinose that induces I-SceI enzyme.
Lac + MBR assays Experimental procedures are as described [53]. The strains to be compared are grown at 37f or two days in M9 medium with 0.1% glycerol and thiamine, then mixed with a 25-fold excess

Chromosomal Tet MBR assays
Experimental procedures are described in [7]. Four cultures of each strain are grown in M9 glycerol liquid medium with 20μg/ml carbenicillin for plasmid maintenance, 50μg/ml proline and with 0.1% glucose to repress P BAD , back-diluted after 12 hours twice and cultured for 84 hours. I-SceI endonuclease is induced by exhaustion of the glucose in the medium. Samples are then plated on complete medium with 0.1% glucose with and without tetracycline to obtain the Tet R mutant frequency, and colonies are counted after one day. Activity of I-SceI was confirmed for all cultures during the experiments by their inability to grow with 0.0001% arabinose in M9 glycerol medium with IPTG ( S2A Fig). I-SceI cutting was confirmed for each strain by the loss of viability when plated on medium without glucose containing 0.001% arabinose (S2B Fig). Error bars on all reported data are the SEM of at least 3 independent experiments. Genes carried in mobile plasmid library plasmids [60] were introduced to strains containing inducible DSB I-SceI cut-sites and enzyme and induced by adding 1mM IPTG to the liquid cultures during growth. A control strain containing an empty overexpression plasmid vector and an I-SceI cutsite and I-SceI enzyme cassette was also similarly treated with IPTG and plated to control for effects of plasmid expression on DSB induction and growth.

Ultraviolet irradiation and MMS treatment
Immediately prior to plating, aliquots of Lac-assay cells in prolonged stationary phase per the Lac MBR assay, were exposed to either UV-C irradiation or MMS. For MMS treatment, 1 mL of starved cell culture was pulse treated with MMS at 5 or 10mM, freshly diluted in water, for 20 minutes at 37˚C. For UV-C irradiation, 1 mL aliquots of each culture were irradiated at either 5 J/m 2 , or 10 J/m 2 using a Stratalinker 2400 UV lamp emitting at 254nm. Viable cell count was determined after MMS and UV treatment to confirm that there was no loss of viability.