SOS-independent bacterial DNA damage responses: diverse mechanisms, unifying function

Abstract

Cells across domains of life have dedicated pathways to sense and respond to DNA damage. These responses are broadly termed as DNA damage responses (DDRs). In bacteria, the best studied DDR is the Save our Soul (SOS) response. More recently, several SOS-independent DDRs have also been discovered. Studies further report diversity in the types of repair proteins present across bacterial species as well as differences in their mechanisms of action. Although the primary function of DDRs is preservation of genome integrity, the diverse organization, conservation, and function of bacterial DDRs raises important questions about how genome error correction mechanisms could influence or be influenced by the genomes that encode them. In this review, we discuss recent insights on three SOS-independent bacterial DDRs. We consider open questions in our understanding of how diversity in response and repair mechanisms is generated, and how action of these pathways is regulated in cells to ensure maintenance of genome integrity.

Introduction

DNA damage is a prevalent threat to genome integrity, and cells across domains of life have evolved dedicated and conserved pathways for repairing or tolerating the same [1]. Sources of DNA damage include endogenous agents such as metabolic by-products or replication errors/stalls and exogenous agents such as radiation, chemicals, as well as toxins and antibiotics secreted into the environment by microbial cells ([2], Table 1). In all cases, cells have mechanisms to sense and respond to DNA damage via a) regulation of cell cycle progression and cell division and b) regulation of repair pathway action [3–7]. While damage repair is important for the faithful propagation of life, some pathways of repair or tolerance can also be sources of mutagenesis [8,9]. Apart from their impact on evolution, such inducible mutagenesis can lead to genetic defects and cancer in human cells as well as antibiotic and stress resistance in bacteria [10–12]. Thus, the regulation of these pathways can have significant impact on cellular adaptation and survival. This becomes particularly relevant in organisms such as bacteria, that live in diverse and fluctuating environments.

Table 1. List of relevant DNA damaging agents and their effects on DNA.

DNA damaging agent	Effect
MNNG	Methylation damage (chiefly 7-methyl guanine, 3-methyl adenine, O6-methyl guanine, O4-methyl thymine, - and methyl phosphotriesters)
MMC	DNA alkylating agent (mono-adducts) and DNA cross-links
Ultraviolet light exposure	Cyclobutane pyrimidine dimers and 6–4 photoproducts
S-adenosyl methionine (SAM)	Methylation damage (chiefly 7-methyl guanine, 3-methyl adenine)
Reactive oxygen species (ROS)	Oxidative damage (8-oxo guanine)

Source: (summarized from [1]).

Cellular responses to DNA damage in bacteria are regulated by specific or generalized transcriptional responses broadly termed as DNA damage responses (DDRs) [5]. The most well-studied bacterial damage response is the SOS response that is activated under many types of DNA damage [13] (Figure 1a). This response is controlled by two genes, recA and lexA) [14]. In an environment devoid of DNA damage, the SOS regulon genes are repressed by LexA, bound upstream of gene promoter regions. RecA activation, via its binding to single-stranded DNA (ssDNA) exposed under DNA damage, acts as a coprotease of LexA [15–17]. This degradation of LexA derepresses the SOS gene promoters, leading to their active transcription. The SOS response encodes for genes involved in diverse repair pathways, including homologous recombination, direct repair, and translesion synthesis [13]. In addition, genes regulating cell cycle progression, including cell division inhibitors, are also expressed as part of this response (such as SulA in Escherichia coli [18], SOS-induced inhibitor of cell division A (SidA) in Caulobacter crescentus [3], and YneA in Bacillus subtilis [19]). Beyond repair and cell division inhibition, genes regulated by the SOS response may play a role in metabolism, apoptosis, toxin production, prophage induction, and pathogenesis [13].

Figure 1. Universal bacterial DDRs.

(a) The SOS response is a highly conserved DDR in bacteria and responds to a variety of DNA damaging agents. The response is regulated by the repressor LexA (bound upstream of SOS-responsive promoters) and RecA. ssDNA-bound RecA triggers the irreversible autocleavage of LexA resulting in the expression of genes involved in DNA damage repair/tolerance and cell division regulation. In addition, toxin–antitoxin systems, metabolic pathways, and prophage genes among others, are also regulated under this response. (b) The Proteasome accessory factor BC (PafBC) response was first identified in Mycobacterium sp. PafB and PafC form a heterodimer that interacts with RNA polymerase to induce PafBC regulon genes under DNA damage. While the precise activation signal for PafBC is unknown, recent work has shown that PafBC–RNA polymerase interaction is potentiated by the presence of ssDNA (shown as a red line in the ‘activated’ PafBC complex) [26]. (c) The DeoR inducer of didA (DriD) response was identified in Caulobacter sp. as an SOS-independent response to DNA damage. DriD forms a homodimer and requires ssDNA (shown as a red line in the ‘activated’ DriD homodimer) for efficient binding to cognate promoters. The exact trigger for the activation of this response is also currently unknown.

In the past few years, multiple SOS-independent responses to DNA damage have also been discovered in bacteria [20–23]. These responses are induced in a manner that is distinct from SOS activation. In this review, we discuss recent insights gained into bacterial DDRs, with a focus on three DDRs that are activated independent of the SOS response. We first briefly describe each DDR pathway (PafBC response, DriD response, and adaptive response to methylation damage). We then discuss common and distinct characteristics of these DDRs, as well as points of overlap (e.g. repair genes that might be co-regulated by multiple DDRs). Toward the end of the review, we speculate on the sources of diversity in bacterial DDRs and the potential impact of the same on genome maintenance. We note here that, although triggered by DNA damage, the direct impact of some of these responses on DNA repair remains to be uncovered.

SOS-independent bacterial DNA damage responses

The PafBC response

The PafBC system was recently described in Mycobacterium sp. as an SOS-independent DDR [21,24] (Figure 1b). PafBC is part of the Proteasome accessory factor ABC (PafABC) operon associated with the Pup proteasomal system. Proteasome accessory factor A is a ligase that attaches a Pup (ubiquitin-like) moiety to proteins that targets them for proteasomal degradation. However, PafB and PafC do not appear to be part of this proteasomal machinery. PafBC belongs to a highly conserved class of bacterial transcription factors that encode for a winged helix-turn-helix domain in their N-terminus and a ‘WYL(Trp (W)–Tyr (Y)–Leu (L))–WCX (WYL protein C-terminal extension)’ domain in their C-terminus. While PafBC-like proteins appear to be predominantly conserved in Actinobacteria, Bacteroidetes, and Firmicutes, this class of proteins can be found in some Proteobacteria as well (Figure 3, an example of the same is discussed in the next section). PafBC forms a regulatory heterodimer (1:1 ratio) that binds to promoters of SOS-independent genes [21] and is required for the expression of majority of all genes induced under mitomycin-C (MMC) damage in Mycobacteria [24,25].

Figure 3. Conservation of DDR pathways across bacterial phyla.

Bacterial genomes were bioinformatically screened for the presence of SOS response (RecA–LexA), Ada response (presence of Ada-like regulator), and PafBC/DriD response (WYL–WCX domain-containing protein) (from [66] as well as analysis conducted for this review). Presence (gray) and absence (white) of the specific DDR pathway are indicated.

Unlike the SOS response, where activator and repressor expression are controlled within the regulon, pafBC genes are not induced under DNA damage [25]. While the specific signals that activate PafBC are presently unknown, a recent study suggests that ssDNA could be a trigger for PafBC activation [26]. In addition, it has been found that gyrase inhibition as well as replication perturbation resulted in PafBC activation [25], supporting the idea that PafBC is indeed an important regulator of the Mycobacterial DDR. Interestingly, although considered independent, the PafBC and SOS responses may also have certain nodes of overlap or cross-talk. An example of this has been found in the context of translesion synthesis, where the imuA’/B genes involved in induced mutagenesis are regulated by both RecA and PafBC [25].

The DriD response

Another DDR discovered outside of the E. coli context is the DriD-regulated response in Caulobacter crescentus [20] (Figure 1c). In Caulobacter, Modell et al. found an SOS-independent cell division inhibitor (damage-induced cell division inhibitor A) DidA that was induced under DNA damage, and most strongly in cells facing double-strand breaks [20]. They further showed that a transcription response activator, DriD, is required for the induction of didA [20]. DriD-regulated didA is induced by a wide range of DNA damaging agents and DriD has been found to bind promoters of several genes (including didA and driD) [27]. DriD also belongs to the class of proteins encoding the ‘WYL–WCX’ motif (with DriD appearing to be conserved in alphaproteobacteria, Figure 3). Similar to PafBC, DriD can induce didA expression without appreciable change in its own levels [20]. It is thus possible that there is post-translational activation of DriD as a transcription factor.

DriD also seems to play a role beyond DDR regulation. More recently, the DriD response has been observed in cells where a gene transfer agent (GTA) cluster has been activated [28]. GTA clusters are prophage-like elements that package linear fragments of DNA [29]. Their production leads to lysis of the host cell. A recipient cell then takes up the packaged DNA via recombination. What is the role for the DriD response in this case? Although not essential for GTA production [28], it is possible that this response plays a role in horizontal gene transfer. This is similar to bacteria such as Streptococcus pneumoniae+ and Bacillus subtilis where an overlap between DDR and natural competence has been reported, with recA expression being regulated by members of the competence machinery instead of LexA [5,30]. It is also possible that the DriD response is induced under these conditions due to increased DNA damage, induced by the activation of the GTA. In support of this possibility, a recent study reported that ssDNA is an allosteric activator of DriD. ssDNA-bound DriD associated with its DNA-binding sites more stably, as well as triggered transcription activation.

Intriguingly, DriD also binds the recA promoter, downstream of the LexA-binding site [27]. This raises the possibility of DriD playing a role in the regulation of the SOS response as well. Consistent with this, Caulobacter cells lacking driD exhibit slower induction of the recA gene [27]. Thus, again similar to PafBC, there could be points of overlap/cross-talk between the SOS and DriD responses as well. Indeed, the direct role of DriD in DNA repair remains to be uncovered. It would additionally be important to understand how conserved the DriD and PafBC systems are and whether points of overlap are common across bacteria that encode multiple DDRs responsive to a variety of damaging agents (we discuss this further in the following sections).

Adaptive response to methylation damage

Apart from the DDRs that respond to multiple DNA damaging agents, cells also encode for a methylation damage-specific response, known as the adaptive response [31] (Figure 2). First described in E. coli, it was found that cells exposed to a sublethal ‘priming’ dose of methylation DNA damage (N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)) were able to withstand a 100-fold higher dose subsequently [32]. The primed (adapted) cells exhibited higher survival and lower mutagenesis as compared with unprimed (nonadapted) cells. This response was shown to be independent of the SOS response [33] and later found to be regulated by the transcription factor ada [34].

Figure 2. SOS-independent response to methylation-specific DNA damage: the adaptive response.

(a) The adaptive response to methylation damage is regulated by the transcription factor Ada, that also has methyltransferase activity. In EcAda takes up aberrant methylation irreversibly from DNA under damage. This activates it as a transcription factor, resulting in the expression of many genes associated with methylation repair (including itself). In this schematic, we focus on N-Ada association with a promoter region. Precise orientation and the role of C-Ada during this stage is not known and hence we represent it as a faded circle. (b–c) Although the adaptive response is conserved, there is significant diversity in the organization of the adaptive response across bacteria. This includes distinct domain organizations of the master regulator of the response (b) as well as differences in the number of homologs found of specific methylation-repair-associated proteins across bacteria (c) (blue denotes presence of a gene with number of homologs in the associated bacterium specified within the box, with no number indicating n = 1, and white denotes the absence of the gene). (c) (Figure 2c adapted from [55], CCA 4.0 license, and includes analysis conducted for this review).

In E. coli, Ada (EcAda) is a 39 kDa protein consisting of two domains, an N-terminal domain (N-Ada) and a C-terminal domain (C-Ada). In an environment devoid of damage, EcAda is maintained at low abundance (0–3 molecules) in most cells and has very low affinity toward the promoters of the adaptive response genes [35].

Under methylation damage, this affinity increases over a hundred-fold, via methylation of N-Ada through a conserved cysteine residue. This methylation has been hypothesized to trigger an electrostatic switch that is responsible for the increased affinity of EcAda toward the adaptive response promoters [36].

There is tight temporal regulation in activation of the SOS and adaptive responses, with SOS being an early response and adaptive response getting activated much later (Figure 4) [37]. In the case of E. coli, the temporal regulation of the two responses appears to be driven by pathway-intrinsic factors and not by potential cross-talk [35,38]. However, it is interesting to note that although both responses act on the same substrates, they have opposite outcomes on repair. The adaptive response results in low mutagenesis, while the SOS response activates mutagenic DNA damage tolerance pathways.

Figure 4. Induction kinetics of DDR pathways. Schematic of induction profiles for the SOS and the adaptive response, as described in E. coli.

(a) SOS response exhibits pulsatile dynamics in the absence of damage. Upon exposure to DNA damage, cells induce SOS response with a ‘short’ lag time attributed to anti-repression-regulated mechanisms. The regulatory circuit for the SOS response is described in Figure 1 as well as the main text. (b) Cells possess variable but low levels of the transcription factor Ada (0–3 molecules) in the absence of damage, resulting in no detectable expression in wild-type growth conditions. Under methylation damage, the adaptive response is induced via activation of Ada, and supersedes the SOS response temporally after a relatively ‘long’ lag time owing to positive autoregulation. There is heterogeneity in induction due to variation in the number of Ada molecules at the single-cell level. Brown cells: response is uninduced; green cells: response is fully induced; brown cells with green dots: green dots indicate the low number of Ada molecules present in cells in the absence of methylation damage.

The adaptive response itself is diverse in its organization across bacteria [31,39,40] (Figure 2b–c). The ada regulon genes differ in their genomic arrangements as well as in terms of the putative effector genes/mechanisms involved in repair. For example, bacteria often possess varying copies of repair proteins (such as ada/ogt methyltransferases) (Figure 2c). Moreover, in some bacteria such as Bacillus sp. and Mycobacterium sp., the two domains of Ada are encoded by separate genes (adaA and adaB), and in Mycobacterium sp., the N-terminus of Ada is also fused with the repair protein, AlkA (Alkylating agent sensitive A) [40–42] (Figure 2b). Thus far, studies have yet to discern the selection pressures that could have influenced adaptive response conservation as well as divergence. Our own analysis reveals that EcAda may be conserved only in ∼17% of sequenced bacterial genomes (searched across a nonredundant database, Kamat et al., in preparation), raising questions about the mechanism of adaptive response action in other organisms and how differences in organization of this response in bacteria may have emerged.

Future perspectives

It is clear that the SOS response represents only one facet of the bacterial DDRs. Some bacteria lack lexA entirely, implying the presence of orthogonal or analogous regulators and/ or responses instead [43]. Indeed, even within the SOS response, there are some critical nuances [44]. RecA, a key regulator of the response, has other essential functions [7], including activation of the translesion synthesis pathway [45], role in natural competence [5], as well as enabling homologous recombination and associated homology search [46,47]. In bacteria with multiple and multitasking response and repair pathways, it hence becomes important to understand the coordination or competition between these systems within the same cell, as this can significantly impact cellular survival as well as genome evolution. How specific are DDRs and what is the extent of their functional overlap? In this direction, we discuss key studies and highlight some outstanding problems below:

Finding common ground: overlapping activation mechanisms and regulatory functions

To appreciate the need for coordination between DDRs, it is important to assess the co-occurrence of multiple responses in the same genome. Computational analysis of the conservation of proteins carrying the WYL–WCX motif (PafBC and DriD) has revealed that proteins carrying this motif are widely conserved across Actinobacteria and Firmicutes. Interestingly, proteins carrying the WYL motif alone are found to be highly conserved in proteobacteria. When we overlay this conservation with that of the SOS (RecA–LexA) and adaptive response (Ada-like proteins) in representative organisms across the bacterial phylogeny, we find that multiple DDRs do co-occur in the same bacterial species (Figure 3). Given that there are several nodes of overlap between these responses at the level of activation as well as downstream function (we briefly discussed some of these nodes of overlap in the previous sections), this co-occurrence underscores the importance of understanding how coordination between DDRs is orchestrated.

For example, at the level of activation, detection of ssDNA appears to be an essential primary step toward mounting a DDR in case of SOS, DriD, and PafBC responses. Similarly, there is also evidence of cross-talk between these responses at the level of the promoters they regulate: both PafBC and DriD bind the recA promoter, with DriD binding upstream of the LexA-binding site [24,27] and similarly, both PafBC and SOS can regulate the expression of the translesion synthesis genes [25,45]. Thus, if these responses do share the same activation signal, how would induction kinetics be regulated in a single cell? It is possible that basal levels of the key regulators differ, resulting in a hierarchy in activation (Figure 4). For example, studies have found that SOS response in E. coli is heterogeneous and inherently leaky [48]. Hence, even in the absence of damage, it spontaneously turns on and such SOS expression follows a pulsatile behavior [38]. On the other hand, Ada is tightly regulated, with only 0–3 molecules of Ada present in the cell in the absence of damage. This results in delayed adaptive response induction, in comparison to the SOS response [35]. It would be insightful to assess the dynamics of the other DDRs in nondamage conditions and how each response is tuned under damage. Such studies will additionally reveal the temporal dynamics of multiple DDRs within the same cell, that could underlie pathway regulation as well.

Keeping it specific

Although there are signs of regulatory cross-talk as well as functional overlap between DDR pathways, there is also some specificity in when a response is activated as well as what type of damage substrate(s) is subsequently repaired [49,50]. As discussed above, specificity in activation can be generated via tight temporal regulation of pathway expression [37,51]. In the presently studied organisms, the SOS-independent pathways appear to be upstream of the SOS response in the regulatory circuit [21,27]. In case of DriD, it is also possible that this response further regulates the timing of SOS response activation, as the DriD-binding site is only a few nucleotides upstream of the LexA box associated with the recA promoter [28].

Along with temporal regulation, it is also possible that the types of DNA damage faced by the cell could shape the presence and action of specific response and repair proteins. The adaptive response to methylation damage is an example of the same [31]. Interestingly, the SOS response itself appears to be diverse across bacterial species, with variation in the number and type of players/mechanisms involved in downstream cell division regulation or repair [44,52]. For example, the SOS regulon of Staphylococcus aureus comprises of only 15 genes, while the recently characterized SOS regulon of Streptomyces venezuelae consists of 97 genes [53,54]. It is possible that further characterization of the repair outcomes of other DDRs could reveal specificity in the repair substrates, distinct impacts on cell survival, and/ or differences in mutagenic outcome of repair. In line with this, Adefisayo et al. have found that SOS response is more essential for UV survival, while PafBC for survival under gyrase inhibition in Mycobacterium sp. [25].

Celebrating diversity

In summary, while mounting a response to DNA damage is ubiquitous, the responses themselves are diverse across bacterial species. Why do bacteria have such diverse organization and function of response and repair pathways? It is tempting to hypothesize that diversity in bacterial genome maintenance mechanisms may be driven by environmental or endogenous factors, such as intermicrobial interactions or genome GC (Guanine-cytosine content) content, that result in varying levels of specific stresses [55–57] (Figure 5). We consider some characteristics here:

Figure 5. What drives diversity in bacterial DDR and repair pathways? We propose some intrinsic and extrinsic factors that could contribute to the same.

(a) Bacteria with different genome characteristics such as location of essential genes with respect to the origin of replication or genome GC content would require congruent DDR pathways. Other differences among bacteria such as cell cycle stage, active metabolic pathways (leading to by-products capable of DNA damage such as SAM or ROS) or costs associated with mutagenic impact of repair could also contribute to the presence or absence of specific DDR pathways. (b) The environment can also affect the type of damage faced by a cell. For example, habitat differences can bring variability in the sources of DNA damage. Interbacterial competition and host defense pathways involve use of methylation-specific DNA damaging agents. Similarly, DNA repair proteins may require nutrients (such as oxygen) as cofactors for function. Presence or scarcity of these nutrients would in-turn play a role in the activity of a specific pathway.

Genome characteristics and cell cycle: The idea that genome characteristics and presence/ absence of repair pathways can be interdependent is exemplified by studies on translesion synthesis [57]. The occurrence of PolV-family UmuDC (UV mutagenesis protein DC) and PolIII-family DnaE2 appears to be correlated with genome GC content [57] and the two classes of polymerases are mutually exclusive, with bacteria such as E. coli (GC content ∼50.8%) encoding UmuDC and other bacteria including Caulobacter sp. (GC content ∼67.2%) having DnaE2 instead [8]. This has also been observed in the case of nonhomologous end-joining (NHEJ) pathway occurrence. The presence of NHEJ proteins is found to be correlated with high genome GC content as well as genome size in bacteria [58,59]. However, how genome characteristics such as GC content affect repair pathway presence needs to be further explored (Figure 5).

Bacterial cell cycle phase may also play a role in determining pathway choice. For example, double-strand break repair in bacteria is accomplished via one of two contrasting pathways, homologous recombination or nonhomologous end joining. Homologous recombination relies on the presence of a duplicate chromosomal copy for faithfully repairing the damage. NHEJ on the other hand involves ligating the two break ends together independent of the presence of a homologous copy. Consistent with this, the NHEJ pathway has been found to be present in bacteria that possess a sporulation phase in their life cycle or spend substantial time in the stationary phase [60].

Costs associated with DDR activation: Another determining factor of DDR organization and conservation could be related to costs associated with the functions of a specific pathway. The SOS response is mutagenic, with its activation leading to mutagenesis even in the absence of damage [61,62]. Could there be such costs associated with other DDRs, resulting in their retention or loss from bacterial genomes? There is also a contradiction to this idea. Although SOS response is inherently leaky and its activation is costly [38,63], it is still largely conserved in bacteria. Hence, despite such costs, pathways could also be retained due to their essentiality under certain stressful conditions. Having multiple responses and associated repair pathways could also confer fitness advantages in cells facing DNA damage. The evolutionary conservation of other known DDRs, their roles beyond damage repair, as well as the cost of misregulated PafBC, DriD, or adaptive responses without DNA damage are yet to be comprehensively uncovered. Future studies in this direction will provide critical insights into the requirement for multiple DDRs and the impact of the same on bacterial genomes.

Environmental effects: Taking the example of environmental selection pressures, a case in point is of AlkB (Alkylating agent sensitive B), an oxidative demethylase that utilizes oxygen as a cofactor in the repair of methylation lesions [64]. Consistent with this property, AlkB has been found to be absent from anaerobic bacteria [65]. Indeed, correlative analysis between the presence or absence of pathways and their environments does suffer from certain drawbacks. Chiefly, classifying parameters such as habitat, aerobicity, and other growth conditions can be difficult. However, these analyses could provide some hints into linking DDR pathway presence and organization with physiological parameters.

Given the intricate relationship between pathways of genome integrity maintenance and mutagenesis, diversity in response and repair capabilities will likely distinctly affect cellular adaptation and survival under DNA damage stress. Thus, gaining better understanding of the factors that govern the organization and conservation of the DDRs across diverse bacterial systems will shed light on how these response and repair mechanisms could be shaped by their cellular and environmental contexts or indeed, be key drivers of genome evolution themselves.

Data Availability

Data will be made available on request.