Induction of only one SOS operon, umuDC, is required for SOS mutagenesis in Escherichia coli

The actions of UmuDC and RecA proteins, respectively in SOS mutagenesis are studied here with the following experimental strategy. We used lexAl (Ind^–) bacteria to maintain all SOS proteins at their basal concentrations and then selectively increased the concentration of either UmuDC or RecA protein. For this purpose, we isolated operator-constitutive mutations o ^c in the umuDC and umuD'C operons and also used the o ₉₈ ^c -recA mutation. The o ₁ ^c -umuDC mutation prevents LexA repressor from binding to the operator and improves the Pribnow box consensus sequence. As a result, 5000 UmuD and 500 UmuC molecules per cell were produced in lexAl bacteria. This concentration is sufficient to restore SOS mutagenesis. The level of RecA protein present in the repressed state promoted full UmuD cleavage. Overproduction of RecA alone did not promote SOS mutagenesis. Increasing the level of RecA in the presence of high concentrations of UmuDC proteins has no further effect on SOS mutgenesis. We conclude that, after DNA damage, umuDC is the only SOS operon that must be induced in Escherichia coli to promote SOS mutagenesis.     

Interactions between epsilon, the proofreading subunit of DNA polymerase III, and proteins involved in the SOS response of Escherichia coli

Summary Epsilon, a fidelity subunit of Escherichia coli DNA Polymerase III, is encoded by dnaQ ⁺. dnaQ49 is a recessive allele that confers temperature-sensitive and saltsuppressible phenotypes for both replication fidelity and viability. SOS mutagenesis in E. coli is regulated by LexA and requires activated RecA (RecA^*) and the products of the umuDC operon. dnaQ49 strains with various recA, lexA and umuDC alleles were constructed to determine if activities induced as part of the SOS response influence epsilon activity. We found: (1) both UmuDC and RecA^* independently enhance the dnaQ49 mutator phenotype, and (2) expression of RecA^* activity in the absence of UmuDC function increases the temperature sensitivity for viability of dnaQ49. These results support the hypothesis that RecA and one or both of the UmuDC proteins interact with the replication complex during SOS mutagenesis.     

Proteolytic processing of MucA protein in SOS mutagenesis: Both processed and unprocessed MucA may be active in the mutagenesis

Summary The mucAB operon carried on plasmid pKM101, which is an analogue of the umuDC operon of Escherichia coli, is involved in UV mutagenesis and mutagenesis induced by many chemicals. Mutagenesis dependent on either the umuDC or mucAB operon requires the function of the recA gene and is called SOS mutagenesis. By treating the cell with agents that damage DNA, RecA protein is activated by conversion into a form (RecA^*) that mediates proteolytic cleavage of the LexA repressor and derepresses the SOS genes including mucAB. Since UmuD protein is proteolytically processed to an active form (UmuD^*) in a RecA^*-dependent fashion, and MucA shares extensive amino acid homology with UmuD, we examined whether MucA is similarly processed in the cell, using antiserum against a LacZ-MucA fusion protein. Like UmuD, MucA protein is indeed proteolytically processed in a RecA^*-dependent fashion. In recA430 strains, MucAB but not UmuDC can mediate UV mutagenesis. However, MucA was not processed in the recA430 cells treated with mitomycin C. We constructed, by site-directed mutagenesis, several mutant mucA genes that encode MucA proteins with alterations in the amino acids flanking the putative cleavage site (Ala25-Gly26). MucA(Cys25) was processed and was as mutagenically active as wild-type MucA; MucA(Asp26) and MucA(Cys25,Asp26) were not processed, and were mutagenically inactive; MucA-(Thr25) was not processed, but was mutagenically as active as wild-type MucA. The mutant mucA gene that encoded the putative cleavage product of MucA was as active as mucA ⁺ in UV mutagenesis. These results raise the possibility that both the nascent MucA and the processed product are active in mutagenesis.     

A umuDC-independent SOS pathway for frameshift mutagenesis.

Summary The chemical carcinogen N-acetoxy-N-2-acetylaminofluorene induces mainly frameshift mutations, which occur within two types of sequences (mutation hot spots): –1 frameshift mutations within contiguous guanine sequences and –2 frameshift mutations within alternating GC sequences such as the NarI and BssHII restriction site sequences. We have investigated the genetic control of mutagenesis at these sequences by means of a reversion assay using plasmids pW17 and pX2, which contain specific targets for contiguous guanine and alternating GC sequences, respectively. Our results suggest that mutations at these hot spot sequences are generated by two different genetic pathways, both involving induction of SOS functions. The two pathways differ both in their LexA-controlled gene and RecA protein requirements. In the mutation pathway that acts at contiguous guanine sequences, the RecA protein participates together with the umuDC gene products. In contrast, RecA is not essential for mutagenesis at alternating GC sequences, except to cleave the LexA repressor. The LexA-regulated gene product(s), which participate in this latter mutational pathway, do not involve umuDC but another as yet uncharacterized inducible function. We also show that wild-type RecA and RecA430 proteins exert an antagonistic effect on mutagenesis at alternating GC sequences, which is not observed either in the presence of activated RecA (RecA^*), RecA730 or RecA495 proteins, or in the complete absence of RecA as in recA99. It is concluded that the –1 mutation pathway presents the same genetic requirements as the pathway for UV light mutagenesis, while the –2 mutation pathway defines a distinct SOS pathway for frameshift mutagenesis.     

Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli

Summary Two multicopy plasmids carrying either the umuDC or the mucAB operon were used to compare the efficiency of UmuDC and MucAB proteins in UV mutagenesis of Escherichia coli K12. It was found that in recA ⁺ uvr ⁺bacteria, plasmid pIC80, mucAB ⁺mediated UV mutagenesis more efficiently than did plasmid pSE117, umuDC ⁺. A similar result was obtained in lexA51(Def) cells, excluding the possibility that this was due to a differential regulation by LexA of the umuDC and mucAB operons. We conclude that some structural characteristic of the UmuDC and MucAB proteins determines their different efficiency in UV mutagenesis. This characteristic could be also responsible for the observation that in the recA430 mutant, pIC80 but no pSE117 can mediate UV mutagenesis. In the recA142 mutant, pIC80 also promoted UV mutagenesis more efficiently than pSE117. In this mutant, the recombination proficiency, the protease activity toward LexA and the mutation frequency were increased by the presence of adenine in the medium. In recA ⁺ uvrB5 bacteria, plasmid pSE117,umuDC caused both an increase in UV sensitivity as well as a reduction in the mutation frequency. These nagative effects resulting from the overproduction of UmuDC proteins were higher in recA142 uvrB5 than in recA ⁺ uvrB5 cells. In contrast, overproduction of MucAB proteins in excision-deficient bacteria containing pIC80 led to a large increase in the mutation frequency. We suggest that the functional differences between UmuDC and MucAB proteins might be due to their different dependence on the direct role of RecA protease in UV mutagenesis.     

Substitution of UmuD' for UmuD does not affect SOS mutagenesis

In order to study the role of UmuDC proteins in SOS mutagenesis, we have constructed new Escherichia coli K-12 strains to avoid i) over-production of Umu proteins, ii) the formation of unwanted mixed plasmid and chromosomal Umu proteins upon complementation. We inserted a mini-kan transposon into the umuD gene carried on a plasmid. The insertion at codon 24 ends protein translation and has a polar effect on the expression of the downstream umuC gene. We transferred umuD24 mutation to the E coli chromosome. In parallel, we subcloned umuD+ umuC+ or umuD' umuC+ genes into pSC101, a low copy number plasmid. In a host with the chromosomal umuD24 mutation, plasmids umuD+ umuC+ or umuD' umuC+ produced elevated resistance to UV light and increased SOS mutagenesis related to a gene dosage of about 3. UV mutagenesis was as high in umuD' umuC+ hosts devoid of UmuD+ protein as in umuD+ umuC+ hosts. UmuD' protein, the maturated form of UmuD, can substitute for UmuD in SOS mutagenesis.     

A new model for SOS-induced mutagenesis: how RecA protein activates DNA polymerase V

In Escherichia coli, cell survival and genomic stability after UV radiation depends on repair mechanisms induced as part of the SOS response to DNA damage. The early phase of the SOS response is mostly dominated by accurate DNA repair, while the later phase is characterized with elevated mutation levels caused by error-prone DNA replication. SOS mutagenesis is largely the result of the action of DNA polymerase V (pol V), which has the ability to insert nucleotides opposite various DNA lesions in a process termed translesion DNA synthesis (TLS). Pol V is a low-fidelity polymerase that is composed of UmuD′₂C and is encoded by the umuDC operon. Pol V is strictly regulated in the cell so as to avoid genomic mutation overload. RecA nucleoprotein filaments (RecA*), formed by RecA binding to single-stranded DNA with ATP, are essential for pol V-catalyzed TLS both in vivo and in vitro. This review focuses on recent studies addressing the protein composition of active DNA polymerase V, and the role of RecA protein in activating this enzyme. Based on unforeseen properties of RecA*, we describe a new model for pol V-catalyzed SOS-induced mutagenesis.     

The frequency of MMS-induced,umuDC-dependent,mutations declines during starvation in Escherichia coli

It has been found that the level of methyl methanesulfonate (MMS)-induced mutation in Escherichia coli is dependent on the level of UmuD(D′)C proteins. The frequency of argE(ochre)→Arg⁺ mutations (which occur predominantly by AT→TA transversions) and Rif^S→Rif^R mutations is much higher when UmuDC or UmuD'C are overproduced in the cell. When MMS-treated bacteria were starved for progressively longer times and hence the expression of mutations delayed, the level of mutations observed progressively declined. This same treatment had no effect on the degree of SOS induction. Examination of plasmid DNAs, isolated from MMS-treated cells, for their sensitivity to the specific endonucleases Fpg and Nth revealed that MMS causes formation of abasic sites, which are repaired during cell starvation. It is assumed that, in non-dividing cells, apurinic sites are mostly repaired by RecA-mediated recombinational repair. This pathway, which is error-free, is compared with the processing pathway in metabolically active cells, where translesion synthesis by the UmuD′₂C-RecA-DNA polymerase III holoenzyme complex occurs; this latter pathway is error-prone.     

Genetic control of the UV-induced SOS mutator effect in single- and double-stranded DNA phages

The SOS hypothesis postulated that the mutator effect on undameged DNA that generates phage-untargeted mutagenesis (UTM) results directly from the mechanism of targeted mutagenesis. RecA protein, which stimulates the cleavage of both the LexA repressor and UmuD protein, and the UmuDC gene products are required for UV-induced targeted mutagenesis. The use of phage λ for analyzing UV-induced mutagenesis has permitted a distinction to be made between the mechanisms of targeted and untargeted mutagenesis, in that the two processes differ with respect to their genetic requirements for recA⁺ and umuDC⁺ genes. In this paper, we show thet (i) proficiency for excision repair is required for UTM in double-stranded DNA phage but not in single-stranded DNA phage; (ii) the umuC function, which is not required for UTM of the double-stranded DNA phage λ, is necessary for untargeted mutagenesis of the single-stranded DNA phages M13 and φX174; (iii) for both single-stranded and double-stranded DNA phage, UV irradiation of the host increases the level of recA730-induced UTM. Our results are also consistent with the interpretation that the expression of untargeted mutagenesis in phage λ and in M13 depends on the polymerase and to a lesser extent on the exonuclease 5′ → 3′, activities of Po1I. These results suggest that the involvement of the RecA and UmuDC proteins may be related to more than the presence of base damage in the DNA substrate.     

The effect of transpiration on selenium uptake and mobility in durum wheat and spring canola

Aims

The objective of this study was to determine the relative importance of transpirational pull, Se speciation, sulfate and species on Se accumulation by plants, in order to determine which of these factors must be considered in the future development of models to predict Se accumulation by plants.

Methods

Seedlings of durum wheat (Triticum turgidum L. var durum cv ‘Kyle’) and spring canola (Brassica napus L. var Hyola 401) were grown hydroponically and exposed to SeO ₄ ^2- (selenate) with or without SO ₄ ^2- (sulfate), or to HSeO ₃ ^- (biselenite) under different transpiration regimes altered through ‘low’ (~50%) or ‘high’ (~78%) relative humidity (RH). Plants were harvested after 0, 8, 16, or 24?h exposures, digested, and analyzed for Se by GFAAS.

Results

Accumulation and distribution of Se by plants is dependent on plant species, Se speciation in the nutrient solution, SO ₄ ^2- competition, and transpiration regimes. Canola accumulated and translocated more Se than wheat. In wheat and canola, the greatest accumulation and translocation of Se occurred when plants were exposed to SeO ₄ ^2- without SO ₄ ^2- compared to solutions of SeO ₄ ^2- with SO ₄ ^2- or HSeO ₃ ^2- . Wheat plants exposed to SeO ₄ ^2- and SO ₄ ^2- had an increased Se accumulation and translocation under increased transpiration rates than when exposed to SeO ₄ ^2- without SO ₄ ^2- or HSeO ₃ ^2- . On the other hand, increases in transpiration increased the translocation of Se to canola shoots when exposed to HSeO ₃ ^- more than any other treatments.

Conclusions

Overall, our results suggest that plant species is the most important factor influencing Se accumulation and translocation, but that these endpoints can be modified by climate and specific soil Se or S content. Models to predict accumulation of Se by plants must consider all of these factors to accurately calculate the mechanisms of uptake and translocation.     

On the presence of sulphate in pituitary lutropin

The presence of sulphate in the carbohydrate of pituitary lutropin from different species has been investigated using a biosynthetic approach. Pituitaries from rats, rabbits, goats, and buffaloes were incubated in the presence of³⁵SO ₄ ^- and the³⁵SO ₄ ^- -labelled proteins in the tissue immunoprecipitated with a well characterized anti-sheep lutropin serum. The incorporation into immunoreactive lutropin was low in the case of rat, rabbit and goat pituitaries while, it was considerable in the case of buffalo pituitaries. Hence further characterization studies were carried out on³⁵SO ₄ ^- -labelled proteins of buffaloes. The physico-chemical, immunological and biological properties of radio-labelled buffalo pituitary material were shown to be similar to those of standard lutropin. Inin vitro conditions of incubations, most of the incorporation of³⁵SO ₄ ^- was observed into tissue lutropin while under similar conditions of incubation, [¹⁴C]-amino acids were found to get incorporated mostly into medium lutropin. The physiologically specific releasing hormone, lutropin-releasing hormone was found to stimulate the release of³⁵SO ₄ ^- -labelled lutropin from the rabbit pituitaries into the medium. These results give indirect evidence that sulphate could be present in pituitary lutropin.     

RecA protein of Escherichia coli has a third essential role in SOS mutator activity.

The DNA damage-inducible SOS response of Escherichia coli includes an error-prone translesion DNA replication activity responsible for SOS mutagenesis. In certain recA mutant strains, in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity. Like UV mutagenesis, SOS mutator activity requires the products of the umuDC operon and depends on RecA protein for at least two essential activities: facilitating cleavage of LexA repressor to derepress SOS genes and processing UmuD protein to produce a fragment (UmuD') that is active in mutagenesis. To determine whether RecA has an additional role in SOS mutator activity, spontaneous mutability (tryptophan dependence to independence) was measured in a family of nine lexA-defective strains, each having a different recA allele, transformed or not with a plasmid that overproduces either UmuD' alone or both UmuD' and UmuC. The magnitude of SOS mutator activity in these strains, which require neither of the two known roles of RecA protein, was strongly dependent on the particular recA allele that was present. We conclude that UmuD'C does not determine the mutation rate independently of RecA and that RecA has a third essential role in SOS mutator activity.     

DinB Upregulation Is the Sole Role of the SOS Response in Stress-Induced Mutagenesis in Escherichia coli

Stress-induced mutagenesis is a collection of mechanisms observed in bacterial, yeast, and human cells in which adverse conditions provoke mutagenesis, often under the control of stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e., are stressed. It is therefore important to understand how stress responses increase mutagenesis. In the Escherichia coli Lac assay, stress-induced point mutagenesis requires induction of at least two stress responses: the RpoS-controlled general/starvation stress response and the SOS DNA-damage response, both of which upregulate DinB error-prone DNA polymerase, among other genes required for Lac mutagenesis. We show that upregulation of DinB is the only aspect of the SOS response needed for stress-induced mutagenesis. We constructed two dinB(o^c) (operator-constitutive) mutants. Both produce SOS-induced levels of DinB constitutively. We find that both dinB(o^c) alleles fully suppress the phenotype of constitutively SOS-“off” lexA(Ind⁻) mutant cells, restoring normal levels of stress-induced mutagenesis. Thus, dinB is the only SOS gene required at induced levels for stress-induced point mutagenesis. Furthermore, although spontaneous SOS induction has been observed to occur in only a small fraction of cells, upregulation of dinB by the dinB(o^c) alleles in all cells does not promote a further increase in mutagenesis, implying that SOS induction of DinB, although necessary, is insufficient to differentiate cells into a hypermutable condition.     

Increased expression of the Escherichia coli umuDC operon restores SOS mutagenesis in lexA41 cells

Summary The lexA41 allele of Escherichia coli encodes a semidefective mutant repressor that is also resistant to RecA facilitated cleavage. Cells harboring the lexA41 allele were found previously to repress only a subset of operons in the SOS regulon. lexA41 cells cannot promote SOS mutagenesis, presumably because one or more operons required for mutagenesis are repressed by this mutant repressor. Using the lac regulatory system to increase the expression of the umuDC operon, we were able to restore mutagenesis in the lexA41 mutant. We conclude that the products of the umuDC operon appear to be uniquely limiting in this mutant.     

SOS repair of 8-methoxypsoralene monoadducts in DNA of lambda bacteriophage and plasmids is mediated by MucA 2 ′ B, but not UmuD 2 ′ C (PolV) polymerase

The light-induced action of 8-methoxypsoralen (8-MOP) on λ phage and plasmids yields monoadducts and interstrand crosslinks. The survival and clear plaque mutation frequency in the phage photosensitized with 8-MOP and irradiated with UV at wavelength >320 nm are increased when the wild-type host (Escherichia coli uvr ⁺) is subjected to UV irradiation (wavelength = 254 nm) prior to phage inoculation. These phenomena are known as “W reactivation” and “W mutagenesis.” It is shown that 8-MOP monoadducts in λ DNA induce clear mutations in the phage inoculated to UV-irradiated excision repair mutants of E. coli only when the error-prone repair is performed by MucA ₂ ^′ B, but not PolV (UmuD ₂ ^′ C) polymerase. The efficiency of the SOS repair (W reactivation) of 8-MOP monoadducts in plasmid and λ phage DNA also only increases with the presence of pKM101 plasmid muc ⁺ in E. coli uvr ^?.     

Directed evolution of GH43 β-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis

Directed evolution of β-xylosidase XylBH43 using a single round of gene shuffling identified three mutations, R45K, M69P, and L186Y, that affect thermal stability parameter K _t ^0.5 by ?1.8 ± 0.1, 1.7 ± 0.3, and 3.2 ± 0.4 °C, respectively. In addition, a cluster of four mutations near hairpin loop-D83 improved K _t ^0.5 by ~3 °C; none of the individual amino acid changes measurably affect K _t ^0.5 . Saturation mutagenesis of L186 identified the variant L186K as having the most improved K _t ^0.5 value, by 8.1 ± 0.3 °C. The L186Y mutation was found to be additive, resulting in K _t ^0.5 increasing by up to 8.8 ± 0.3 °C when several beneficial mutations were combined. While k _cat of xylobiose and 4-nitrophenyl-β-d-xylopyranoside were found to be depressed from 8 to 83 % in the thermally improved mutants, K _m, K _ss (substrate inhibition), and K _i (product inhibition) values generally increased, resulting in lessened substrate and xylose inhibition.     

The genetic requirements for UmuDC-mediated cold sensitivity are distinct from those for SOS mutagenesis.

The umuDC operon of Escherichia coli, a member of the SOS regulon, is required for SOS mutagenesis. Following the posttranslational processing of UmuD to UmuD' by RecA-mediated cleavage, UmuD' acts in concert with UmuC, RecA, and DNA polymerase III to facilitate the process of translesion synthesis, which results in the introduction of mutations. Constitutive expression of the umuDC operon causes an inhibition of growth at 30 degrees C (cold sensitivity). The umuDC-dependent physiological phenomenon manifested as cold-sensitive growth is shown to differ from SOS mutagenesis in two respects. Intact UmuD, the form inactive in SOS mutagenesis, confers a significantly higher degree of cold sensitivity in combination with UmUC than does UmuD'. In addition, umuDC-mediated cold sensitivity, unlike SOS mutagenesis, does not require recA function. Since the RecA protein mediates the autodigestion of UnmD to UmuD', this finding supports the conclusion that intact UmuD is capable of conferring cold sensitivity in the presence of UmuC. The degree of inhibition of growth at 30 degrees C correlates with the levels of UmuD and UmuC, which are the only two SOS-regulated proteins required to observe cold sensitivity. Analysis of the cellular morphology of strains that exhibit cold sensitivity for growth led to the finding that constitutive expression of the umuDC operon causes a novel form of sulA- and sfiC-independent filamentation at 30 degrees C. This filamentation is observed in a strain constitutively expressing the single, chromosomal copy of umuDC and can be suppressed by overexpression of the ftsQAZ operon.     

NO 3 − transport across the plasma membrane of Arabidopsis thaliana root hairs: Kinetic control by pH and membrane voltage

High-affinity nitrate transport was examined in intact root hair cells of Arabidopsis thaliana using electrophysiological recordings to characterise the response of the plasma membrane to NO ₃ ^? challenge and to quantify transport activity. The NO ₃ ^? -associated membrane current was determined using a three-electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in the roots of seedlings grown in the absence of a nitrogen source, but only 4–6 days postgermination. In 6-day-old seedlings, additions of 5–100 μm NO ₃ ^? to the bathing medium resulted in membrane depolarizations of 8–43 mV, and membrane voltage (V _m) recovered on washing NO ₃ ^? from the bath. Voltage clamp measurements carried out immediately before and following NO ₃ ^? additions showed that the NO ₃ ^? -evoked depolarizations were the consequence of an inward-directed current that appeared across the entire range of accessible voltages (?300 to +50 mV). Both membrane depolarizations and NO ₃ ^? -evoked currents recorded at the free-running voltage displayed quasi-Michaelian kinetics, with apparent values for K_m of 23 ± 6 and 44 ± 11 μm, respectively and, for the current, a maximum of 5.1 ± 0.9 μA cm^?2. The NO ₃ ^? current showed a pronounced voltage sensitivity within the normal physiological range between ?250 and ?100 mV, as could be demonstrated under voltage clamp, and increasing the bathing pH from 6.1 to 7.4–8.0 reduced the current and the associated membrane depolarizations 3- to 8-fold. Analyses showed a well-defined interaction between the kinetic variables of membrane voltage, pH_o and [NO ₃ ^? ]_o. At a constant pH_o of 6.1, depolarization from ?250 to ?150 mV resulted in an approximate 3-fold reduction in the maximum current but a 10% rise in the apparent affinity for NO ₃ ^? . By contrast, the same depolarization effected an approximate 20% fall in the K_m for transport as a function in [H⁺]_o. These, and additional characteristics of the transport current implicate a carrier cycle in which NO ₃ ^? binding is kinetically isolated from the rate-limiting step of membrane charge transit, and they indicate a charge-coupling stoichiometry of 2(H⁺) per NO ₃ ^? anion transported across the membrane. The results concur with previous studies showing a high-affinity NO ₃ ^? transport system in Arabidopsis that is inducible following a period of nitrogen-limiting growth, but they underline the importance of voltage as a kinetic factor controlling NO ₃ ^? transport at the plant plasma membrane.     

The appearance of the UmuD'C protein complex in Escherichia coli switches repair from homologous recombination to SOS mutagenesis

The process of SOS mutagenesis in Escherichia coli requires (i) the replisome enzymes, (ii) RecA protein, and (iii) the formation of the UmuD'C protein complex which appears to help the replisome to resume DNA synthesis across a lesion. We found that the UmuD'C complex is an antagonist of RecA-mediated recombination. Homologous recombination in an Hfr x F^- cross decreased as a function of the UmuD'C cell concentration; this effect was challenged by increasing RecA concentration. Recombination of a u.v.-damaged F-lac with the lac gene of an F^- recipient was reduced by increasing the UmuD'C concentration while lac mutagenesis increased, showing an inverse relationship between recombination and SOS mutagenesis. We explain our data with the following model. The kinetics of appearance of the UmuD'C complex after DNA damage is slow, reaching a maximum after an hour. Within that period, excision and recombinational repair have had time to occur. When the UmuD'C concentration relative to the number of residual RecA filaments, not resolved by recombinational repair, becomes high enough, UmuD'C proteins provide a processive factor for the replisome to help replication bypass and repel the standing RecA filament. Thus, at a high enough concentration, the UmuD'C complex will switch repair from recombination to SOS mutagenesis.