SOS RESPONSE REGULATION
The SOS response is initiated by accumulation of single‐stranded (ss) DNA during replication of DNA containing lesions (Sassanfar and Roberts, 1990). Single‐stranded DNA is generated when DNA polymerase stalls at a template lesion while the replicative helicase continues unwinding DNA (Higuchi et al., 2003; Pagès and Fuchs, 2003). Recent studies indicate that the replisome skipping over template lesions and repriming to allow reinitiation of DNA synthesis downstream of a block leaving behind a single‐stranded DNA gap is possible during both leading and lagging strand synthesis (Yeeles and Marians, 2013; Indiani and O’Donnell, 2013; Gabbai et al., 2014). It should be mentioned that during horizontal gene transfer ssDNA is also transiently present and both transformation and conjugative plasmid DNA transfer induce the SOS response (Baharoglu et al., 2012). However, a number of conjugative plasmids encode the PsiB protein, which inhibits the induction of SOS during conjugation (Bagdasarian et al., 1986).
Two proteins play key roles in the regulation of the SOS response: LexA and RecA. LexA protein is composed of two domains separated by a short flexible linker: N‐terminal DNA binding domain (NTD), and a C‐terminal catalytic domain (CTD) with a serine‐lysine catalytic dyad. CTD is also responsible for homodimerization of LexA (Luo et al., 2001; Zhang et al., 2010). During normal growth, the LexA dimer acts as a transcriptional repressor for genes belonging to the SOS regulon by binding to a specific operator sequence (the SOS box) in their promoter region (Walker, 1984). RecA acts as a co‐protease to stimulate self‐cleavage of LexA as well as other related proteins, such as phage repressors (λ, φ80, P22, 434), UmuD protein and its homologs (Walker, 1984). RecA binds the ssDNA, and in the presence of a nucleoside triphosphate converts to an activated form (RecA* nucleoprotein filament) (Patel et al., 2010, reviewed in Goodman et al., 2016; Jaszczur et al., 2016). RecA* stimulates self‐cleavage of LexA at a specific Ala84‐Gly85 bond near the middle of the protein, thereby de‐activating LexA (Little, 1991), lowering its affinity for the DNA and exposing residues that target LexA for ClpXP and Lon protease degradation (Neher et al., 2003). As a result, the pools of LexA protein begin to decrease, leading to derepression of SOS genes (Little and Mount, 1982). Figure Figure11 schematically presents the SOS induction process.
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The SOS response entails increased expression of over 50 genes that carry out diverse functions in response to DNA damage, including excision repair, homologous recombination, translesion DNA replication, and cell division arrest; some of these genes encode proteins of unknown function (see Table Table1).1). In addition, other genes can be upregulated following DNA damage but are believed to be independent of LexA (Fernández De Henestrosa et al., 2000; Courcelle et al., 2001, comprehensively analyzed in Cohen et al., 2008). Not all genes belonging to the SOS regulon are induced at the same time and to the same level. The response is precisely timed and synchronized according to the amount of damage and the time elapsed since the damage was detected; selective derepression of certain genes might occur in response to even minor endogenous DNA damage, while other genes will be expressed only upon drastic DNA damage and a persistent inducing signal (Fernández De Henestrosa et al., 2000; Courcelle et al., 2001; Quillardet et al., 2003). Mostly error‐free repair and maintenance processes characterize the early phase of SOS. The first genes induced are the uvr genes for excision of damaged nucleotides, followed by recA and other homologous recombination protein coding genes (ruvAB, recN). Next are polB and dinB encoding DNA polymerase II and DNA polymerase IV, respectively. The division inhibitor SulA is also induced to give the bacterium time to complete the repairs. Finally, if the damage was extensive and not fully repaired, the error‐prone DNA polymerase V (encoded by umuC and umuD genes) is induced, causing elevated mutation levels but allowing for continuous replication and cell survival (Courcelle et al., 2001; Henrikus et al., 2018b).
The level, timing, and duration of induction of different LexA‐regulated genes depends on the strength of different SOS boxes, their number and location relative to the target promoter, and promoter strength. The consensus SOS box sequence is a perfect palindrome: TACTG(TA)5CAGTA. The deviation of an SOS box from the consensus is characterized by the heterology index (HI). The higher the HI value, the lower the affinity of the LexA repressor to the sequence (Lewis et al., 1994; Fernández De Henestrosa et al., 2000).
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Importantly, lexA itself is also a SOS gene. The constant production of LexA during the SOS process ensures that as soon as DNA repair occurs, the disappearance of the inducing signal will allow LexA to reaccumulate and repress the SOS genes (Walker, 1984). In an uninduced cell, roughly 1,300 molecules of LexA are present (Sassanfar and Roberts, 1990). In addition to this loop mechanism, several SOS genes seem to be involved in more precise modulation of the SOS response. For instance, RecX and DinI affect the stability of the RecA filament, thereby influencing the response time and recovery rate of the system (Lusetti et al., 2004a). The RecX protein is known to be a very active RecA inhibitor; it can suppress various RecA activities including ATP hydrolysis, coprotease, and DNA strand exchange reaction at a concentrations hundreds of times smaller than that of the RecA itself (Stohl et al., 2003). It blocks the extension of the RecA filaments (Drees et al., 2004) and promotes RecA filament disassembly (Ragone et al., 2008). Based on the structure of RecX protein complex with the presynaptic RecA filament, Yakimov et al. (2017) designed a novel peptide inhibitor with highly stable α‐helical structure capable not only of inhibiting the RecA protein activities in vitro but also of suppressing the E. coli SOS response in vivo. A peptide binding the RecA filament groove may also provide a valuable tool for studying RecA interactions with a wide range of proteins interacting with RecA filaments. Function of DinI depends on the concentration of the protein. At concentration stoichiometric with the concentration of RecA, it acts mainly as a positive modulator of RecA function, stabilizing the RecA filament (Lusetti et al., 2004a, b). At high concentrations, it has an opposite effect on RecA ‐ it inhibits both the ability of RecA to induce cleavage of UmuD as well as its recombinase activity (Yasuda et al., 1998, 2001).
The umuDC operon was also identified as a key contributor maintaining the temporal precision of the SOS response. The uncleaved UmuD protein and UmuC delay the recovery of DNA replication after DNA damage to allow additional time for accurate repair systems to process the damage (Opperman et al., 1999). Using time‐lapse fluorescence microscopy Friedman et al. (2005) investigated the dynamics of SOS response in individual living cells. They showed that the products of the umuDC operon are involved in modulating SOS expression by generating discrete activation pulses, whose number increases with the level of DNA damage. McCool et al. (2004) using fluorescent microscopy showed that within the population of DNA metabolism mutants with high basal levels of SOS expression exist subpopulations of cells with various levels of SOS expression. This suggests that in cells where multiple pathways are available to process DNA intermediates the choice of pathway may affect SOS genes expression levels.
Certain stimuli can indirectly generate the SOS‐inducing signal by activation of endogenous DNA damage mechanisms rather than by direct DNA damage, which suggests that SOS might be involved in adaptation to various types of stresses (reviewed by Aertsen and Michiels, 2006). Kubiak et al. (2017) have developed a system for inducible control over the SOS pathway independent of DNA damage and RecA* in vivo. They engineered a LexA variant with abolished self‐cleavage, but with optimized recognition site for TEV protease placed within the flexible linker between the C‐ and N‐terminal domains. This opens the possibility of addressing questions related to SOS function independently of DNA damage.
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