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Copyright _ 2009 by the Genetics Society of America
DOI: 10.1534/genetics.109.100735
DinB Upregulation Is the Sole Role of the SOS Response innStress-Induced Mutagenesis in Escherichia coli

Rodrigo S. Galhardo,* Robert Do,* Masami Yamada,Errol C. Friedberg,P. J. Hastings,*
Takehiko Nohmiand Susan M. Rosenberg*,§,1

*Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030-3411, ‡Department of Pathology, University
of Texas Southwestern Medical Center, Dallas, Texas 5390-9072, †Division of Genetics and Mutagenesis, National Institute of Health
Sciences, Tokyo 158-8501, Japan and §Departments of Biochemistry and Molecular Biology and Molecular Virology
and Microbiology, The Dan L. Duncan Cancer Center, Baylor College of Medicine,
Houston, Texas 77030-3411
Manuscript received January 12, 2009
Accepted for publication February 26, 2009


Abstrak

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 DNAdamage
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(oc) (operator-constitutive) mutants. Both
produce SOS-induced levels of DinB constitutively. We find that both dinB(oc) 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(oc) 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.


GENOMIC stability and mutation rates are tightlyregulated features of all organisms. Understanding how cells regulate mutation rates has importantimplications for evolution, cancer progression and chemotherapy
resistance, aging, and acquisition of antibioticresistance and evasion of the immune system by
pathogens, all processes driven by mutagenesis and all ofwhich occur during stress.
Stress-induced mutagenesis refers to a group of relatedphenomena in which cells poorly adapted to their
environment (i.e., stressed) increase mutation ratesas part of a regulated stress response (reviewed by
Galhardo et al. 2007). Abundant examples, particularlyin microorganisms, show the induction of specific pathwaysof mutagenesis in response to stresses. The types ofgenetic alteration induced by stress include base substitutions,small deletions and insertions, gross chromosomal
rearrangements and copy-number variations, andmovement of mobile elements. These various pathwaysrequire the functions of different sets of genes andproteins. Thus, there appear to be multiple molecular mechanisms of stress-inducible mutagenesis that operatein different organisms, cell types, and growthinhibiting stress conditions.
However, a common theme in the many mechanisms
of stress-inducible mutagenesis described to date is therequirement for the function of one or more cellular
stress responses. Starvation stress-induced mutagenesisin Bacillus subtilis requires the comK regulatory gene that
controls the stress response that in turn allows competence for natural transformation in response to starvation
(Sung and Yasbin 2002). The RpoS-controlledgeneral or starvation stress response is required forstarvation-induced excisions of phage Mu in Escherichia coli (Gomez-Gomez et al. 1997), for base-substitution mutagenesis in aging E. coli colonies (Bjedov et al.2003), for starvation-induced point mutations (Saumaa
et al. 2002) and transpositions (Ilves et al. 2001) in Pseudomonas putida, and for starvation-induced gene amplification (Lombardo et al. 2004) and frameshift  mutagenesis (Layton and Foster 2003; Lombardo
et al. 2004) in the E. coli Lac assay, described in more
detail below. The SOS DNA-damage stress response isrequired for the stress-induced frameshift mutagenesis
in the E. coli Lac assay discussed below, for E. coli
mutagenesis in aging colonies (Taddei et al. 1997), for ciprofloxacin (antibiotic)-induced resistance mutagenesis
(Cirz et al. 2005), and for mutagenesis conferring
resistance to bile salts in Salmonella (Prieto et al. 2006). The stringent response to amino-acid starvation is required
for a transcription-associated mutagenesis in E. coli that targets stringent-response-controlled genes(Wright et al. 1999) and for amino-acid-starvationinduced mutagenesis in B. subtilis (Rudner et al. 1999).Two different stress responses to hypoxia in humancancer cells also increase mutagenesis. One does so by
specific downregulation of mismatch-repair genes (Mihaylova et al. 2003; Koshiji et al. 2005; Bindra
and Glazer 2007). The other is postulated to promote genome rearrangement by its demonstrated downregulation
of RAD51 andBRCA1 functions required for high-fidelity repair of DNA double-strand breaks (DSBs)
(Bindra et al. 2004). These stress responses exert temporal control or restriction of mutagenesis, which
favors genomic stability when cells and organisms are well adapted to their environments (i.e., not stressed)
and increases mutagenesis, potentially accelerating evolution, specifically during stress when cells are maladapted
to their environments. Except for the human examples, the ways by which the stress responses upregulate
mutagenesis are mostly not understood.We focus here on how a stress response controls mutagenesis in an  E. coli model system.

Stress-induced mutagenesis is perhaps best understood in the E. coli model system. A widely used assay
system uses a 11 frameshift allele of a lacIZ fusion gene located in the F9128 plasmid in cells with a deletion of
the chromosomal lac genes (Cairns and Foster 1991). When these cells are plated on lactose minimal medium,
a few Lac1 revertant colonies are observed. Many of these arise from spontaneous generation-dependent
mutations that occur during growth of the culture. Prolonged incubation of these plates results in the
continuous accumulation of additional Lac1 revertants, which arise through two mechanisms, both different
from the mechanisms that produce the generationdependent mutants (reviewed by Galhardo et al. 2007).

First, within the first few days, most of the Lac1 colonies are ‘‘point mutants’’ that possess a compensatory
_1 frameshift mutation in the lacIZ gene (Foster Trimarchi 1994; Rosenberg et al. 1994). Cells that
carry these mutations also carry increased numbers of secondary unselected mutations in other genomic regions,
whereasmost Lac_ cells starved on the same plates do not, indicating that a subpopulation of the cells undergoes genomewide hypermutation (Torkelson et al. 1997; Rosche and Foster 1999; Godoy et al. 2000). Therefore, a subset of the starved cells experiences increased mutagenesis when compared with the
majority of the cells. Hereafter we refer to this subpopulation as ‘‘hypermutable.’’ This hypermutable cell
subpopulation (HMS) appears to be important to the
formation of most or all of the Lac1 stress-induced mutants (Gonzales et al. 2008). The hypermutable state is
transient, ceasing after growth impairment is ended and growth resumes (Longerich et al. 1995; Torkelson
et al. 1997; Rosenberg et al. 1998; Rosche and Foster 1999; Godoy et al. 2000).

Second, longer incubation also results in the formation  of a significant proportion of lac-amplified colonies in which the leaky lacIZ allele is amplified to 20–50 tandem copies, which produce sufficient enzyme activity to allow growth on lactose (Hastings et al. 2000; Powell and Wartell 2001; Kugelberg et al. 2006; Slack et al. 2006). In summary, E. coli cells may either increase pointmutation rates or undergo extensive genomic rearrangement in response to a growth-limiting environment.
Both of these processes require induction of the general or starvation stress response controlled by RpoS
(Lombardo et al. 2004). Point mutagenesis, but not amplification, also requires induction of the SOS DNAdamage
stress response (Cairns and Foster 1991; McKenzie et al. 2000, 2001). In this article, we focus
on the role of the SOS response in the mechanism of stress-induced point mutagenesis. SeeHastings (2007)
for a review of the mechanisms of stress-induced amplification and genome rearrangement.
The molecular mechanism of point mutagenesis in the Lac system is now considerably well understood. It entails a switch from the normally high-fidelity DNA synthesis associated with recombination-dependent double-strand-break repair to an error-prone synthesis specifically under stress (Ponder et al. 2005). Several
genetic requirements are known for stress-induced point mutagenesis, including DNA-recombination functions
(Harris et al. 1994, 1996; Foster et al. 1996; He et al. 2006) in addition to the genes required for induction of
the SOS DNA-damage response (Cairns and Foster 1991;McKenzie et al. 2000) and the sS (RpoS) general/
starvation stress-response (Layton and Foster 2003; Lombardo et al. 2004) regulons, and the dinB gene
encoding DNA polymerase (Pol) IV (McKenzie et al. 2001).
DinB is the founding member of the most widespread subfamily of Y-family specialized DNA polymerases, with orthologs in bacteria, archaea, and eukaryotes, including humans (reviewed by Nohmi 2006). DinB/ Pol IV can perform high-fidelity translesion DNA synthesis (TLS) across a number of different DNA lesion substrates ( Jarosz et al. 2006; Bjedov et al. 2007; Yuan et al. 2008). However, this enzyme shows a significant
error rate when copying undamaged DNA templates (Kobayashi et al. 2002). Some mutations in DinB can abolish its TLS activity, without interfering with the mutator phenotype caused by overexpression of DinB,
suggesting that mutagenesis and TLS are independent activities of Pol IV (Godoy et al. 2007). Eighty-five
percent of the stress-induced Lac1 point mutations generated in the nongrowing cells arise in a DinBdependent manner (McKenzie et al. 2001).
The dinB gene is under the control of the SOS response, which upregulates its transcription 10-fold
(Kim et al. 2001). Additionally, the alternative s (transcription) factor sS (RpoS), which is responsible for the
general stress response, upregulates dinB expression transcriptionally by 2- to 3-fold upon entry into stationary
phase (Layton and Foster 2003). Proteins such as Ppk (Stumpf and Foster 2005) and the chaperones
GroEL (Layton and Foster 2005), RecA, and UmuD (Godoy et al. 2007) all seem to modulate DinB activity.
An interesting in vivo role of DinB is SOS untargeted mutagenesis of phage l (Kim et al. 1997). In it, _1
frameshift mutations in runs of G’s are generated, similarly to the predominant mutations detected in
the lac gene during stress-induced mutagenesis (Foster and Trimarchi 1994; Rosenberg et al. 1994). On the
other hand, DinB has no effect on the spontaneous mutation rate in growing cells (McKenzie et al. 2001,
2003; Kuban et al. 2004; Wolff et al. 2004). DinB is implicated as the DNA polymerase that, only during the
stress responses, makes DSB-repair-associated errors that become stress-induced point mutations (Ponder
et al. 2005).
The role of the SOS response in controlling mutagenesis in the Lac assay is a complex issue because several SOS-controlled genes are required for the process. dinB, recA, ruvA, and ruvB are all required for
mutagenesis (Cairns and Foster 1991; Harris et al. 1994, 1996; Foster et al. 1996; McKenzie et al. 2001; He
et al. 2006) and are all upregulated by SOS (Courcelle et al. 2001). Also, the F-encoded psiB gene exerts a negative effect onmutagenesis in SOS-derepressed cells (McKenzie et al. 2000) and is thought to inhibit SOS
induction and RecA (reviewed by Cox 2007).We sought to determine whether the requirement for induction of
the SOS response in stress-induced mutagenesis reflects a need for upregulation solely of dinB or whether any
other gene(s) is required at SOS-induced levels. We present evidence below that indicates, first, that DinB is
the only SOS-controlled gene required at induced levels for efficient stress-induced point mutagenesis and,
second, that, although SOS-induced levels of DinB are required, they are not sufficient to differentiate cells into a hypermutable condition.

MATERIALS AND METHODS
Bacterial strains, plasmids, and media: The bacterial strains used in this work are shown in Table 1. dinB(oc) alleles were constructed as described below. Other strains were constructed using P1-mediated transduction as described (Miller 1992).
The antibiotics used were as follows: ampicillin, 100 mg/ml; chloramphenicol, 25 mg/ml; tetracycline, 10 mg/ml; kanamycin,
30 mg/ml; and rifampicin, 40 mg/ml. 5-Bromo-4-chloro-3- indolyl-b-d-galactoside (X-gal) was used at 40 mg/mL. M9
minimal medium (Miller 1992) was supplemented with 10 mg/ml of vitamin B1 and either 0.1% glycerol or 0.1%
lactose. Luria–Bertani–Herskowitz (LBH) medium was used as described by Torkelson et al. (1997).
The plasmids used in this study are listed in Table 2. Plasmids containing PdinBlacZ fusions used in b-galactosidase
assays for gene expression analysis were constructed by amplification of the dinB promoter (from bases _432 to _2 of dinB) with primers 59-TCGGCTGAATTCTGTTCGA CTCGCTCGATAAT-39 and 59-CGGTACAAGCTTGCTCACCT CTCAACACTGGT-39 and by cloning into the pFZY plasmid (Koop et al. 1987) using the EcoRI and HindIII sites introduced in the primers. The dinB promoter was amplified from strain SMR4562 and cloned into pFZY to generate plasmid pPdinB
and amplified from strains SMR10308 and SMR10309 to generate the plasmids pPdinBOC1 and pPdinBOC2, respectively.
Construction of the dinB(oc) alleles and strains bearing them: We created each of two mutations predicted from previous work on other SOS genes (Friedberg et al. 2005) to inactivate the predicted LexA-binding site in dinB (Figure
1A). The Lac assay strains carry two copies of dinB, one in the chromosome and one in F9128 (discussed in the results).
The constructions required several steps as below. Primer sequences are given in the supporting information, File S1.
First, we linked the cat selectable marker with dinB. We chose to put a selectable marker in the lafU (formerly known as mbhA) gene, which is present immediately upstream from the 59-end of dinB. The FRTcatFRTcassette was amplified from pKD3 (Datsenko and Wanner 2000) using primers CatupdinB-F and CatupdinB-R. The product was used to obtain SMR4562 recombinants containing the lafUTFRTcatFRT insertion (allele DlafU2TFRTcatFRT), using short-homology recombination as described (Datsenko and Wanner 2000). One recombinant containing the DlafU2TFRTcatFRT in the F9 plasmid was selected. This strain (SMR10292) was used to amplify the DlafU2TFRTcatFRT-dinB1 region by PCR using primers
CatupdinB-F and dinBcatnock-R. This product was used as a template for PCR-mediated site-directed mutagenesis, altering the dinB promoter.
Next we constructed a DlafU-dinB deletion strain to be used as a recipient for allelic replacement with the site-directed dinB-mutant genes linked to DlafU2TFRTcatFRT.We created a FC36-derivative containing a deletion encompassing the 39 half of DlafU and the whole dinB gene using primers kandinBchrom-F and DinBRCAT to amplify FRTKanFRT from pKD13 (Datsenko and Wanner 2000). The products were used for short-homology recombination in the FC36 background, creating strain SMR10299. A similar deletion in the same region in the F9 plasmid was created by short-homology recombination in SMR4562, using FRTcatFRT amplified from pKD3 with primers CatupdinB-F and DinBRCAT. Location of the deletion in the F9 plasmid was confirmed by the ability to conjugate the cat gene conferring chloramphenicol resistance. The cat gene was removed by FLP-mediated site-specific recombination using the pCP20 plasmid (Datsenko and Wanner 2000). The resulting F9128 DlafU-dinBTFRT [allele D(lafU-dinB)2096(TFRT)] was mated into strain SMR10299, creating strain SMR10303 {SMR4562 D(lafU dinB)2097(TFRTKanFRT) [F9 D(lafU-dinB)2096(TFRT)]}. This strain was used as a recipient for allelic replacement using the site-directed dinB mutants produced by PCR with the DlafU2TFRTcatFRTdinB fragment as a template. The sequence of the promoter and coding sequence of the dinB gene from the KanR CamR recombinants was determined by PCR and DNA sequencing to



TABLE 1
Bacterial strains used in this study



Name
Relevant genotype

Reference or source


FC29
FC40
FC231
SMR868
SMR4562
SMR5400
SMR9436
SMR5889
SMR10292
SMR10299
SMR10303
SMR10304

SMR10306

SMR10308
SMR10309
SMR10310
SMR10311
SMR10314
SMR10760
SMR10761
SMR10762
SMR10766
SMR10767
SMR10768
SMR10838
SMR10839
SMR10840
SMR10841
SMR10842
SMR10843
SMR11023
SMR11024
SMR11026
SMR11027

D(lac-proB)XIII ara thi [F9 D(lacI-lacZ)]
D(lac-proB)XIII ara thi Rif R [F9 lacI33VlacZ proAB1]
FC40 lexA3(Ind_)
FC40 lexA3(Ind_)
Identical to FC40, independent construction
SMR4562 sulA211 lexA51(Def) DpsiBTcat
SMR4562 DruvCTFRTKanFRT
SMR4562 DdinB50TFRT [F9 DdinB50TFRT]
SMR4562 [F9 lafU2TFRTcatFRT]
FC36 D(lafU-dinB)2097(TFRTKanFRT)
SMR4562 D(lafU-dinB)2097(TFRTKanFRT) [F9 D(lafU-dinB)2096(TFRT)]
SMR4562 D(lafU-dinB)2097(TFRTKanFRT) [F9 lafU2TFRTcatFRT dinBo-21(oc)]
SMR4562 D(lafU-dinB)2097(TFRTKanFRT) [F9 lafU2TFRTcatFRT dinBo-22(oc)]
SMR4562 [F9 lafU2TFRTcatFRT dinBo-21(oc)]
SMR4562 [F9 lafU2TFRTcatFRT dinBo-22(oc)]
SMR868 [F9 lafU2TFRTcatFRT dinBo-21(oc)]
SMR868 [F9 lafU2TFRTcatFRT dinBo-22(oc)]
SMR868 [F9 lafU2TFRTcatFRT]
FC231 [F9 lafU2TFRTcatFRT]
FC231 [F9 lafU2TFRTcatFRT dinBo-21(oc)]
FC231 [F9 lafU2TFRTcatFRT dinBo-22(oc)]
SMR4562 DruvCTFRTKanFRT [F9 lafU2TFRTcatFRT]
FC231 DruvCTFRTKanFRT [F9 lafU2TFRTcatFRT dinBo-21(oc)]
FC231 DruvCTFRTKanFRT [F9 lafU2TFRTcatFRT]
SMR4562 [pPdinB]
SMR4562 [pPdinBOC1]
SMR4562 [pPdinBOC2]
SMR5400 [pPdinB]
SMR5400 [pPdinBOC1]
SMR5400 [pPdinBOC2]
SMR4562 [F9 lafU2TFRTcatFRT DyafNOPTFRTKanFRT]
SMR4562 [F9 lafU2TFRTcatFRT DyafNOPTFRTKanFRT dinBo-21(oc)]
FC231 [F9 lafU2TFRTcatFRT DyafNOPTFRTKanFRT]
FC231 [F9 lafU2TFRTcatFRT DyafNOPTFRTKanFRT dinBo-21(oc)]

Cairns and Foster (1991)
Cairns and Foster (1991)
Cairns and Foster (1991)
McKenzie et al. (2000)
McKenzie et al. (2000)
McKenzie et al. (2000)
Magner et al. (2007)
McKenzie et al. (2001)
This study
This study
This study
This study

This study

SMR4562 3 P1(SMR10304)
SMR4562 3 P1(SMR10306)
SMR868 3 P1(SMR10304)
SMR868 3 P1(SMR10306)
SMR868 3 P1(SMR10292)
FC231 3 P1(SMR10292)
FC231 3 P1(SMR10304)
FC231 3 P1(SMR10306)
SMR10292 3 P1(SMR9436)
SMR107613 P1(SMR9436)
SMR10760 3 P1(SMR9436)
This study
This study
This study
This study
This study
This study
This study
This study
FC231 3 P1(SMR11023)
FC231 3 P1(SMR11024)



ensure that the desired mutation was introduced and that no other mutation in dinB was generated inadvertently by
PCR. One recombinant containing the dinBo-21(oc) mutation (SMR10304) and one containing the dinBo-22(oc) mutation
(SMR10306) were chosen. Those strains were used as P1 donors of DlafU2TFRTcatFRT dinBo-21(oc) and DlafU2T
FRTcatFRT dinBo-22(oc), respectively, to transduce the din- B(oc) alleles to all the genetic backgrounds of interest,
including SMR4562 and strains FC231 and SMR868 carrying lexA3(Ind_).
Deletion of the yafNOP genes in the dinB operon was performed using short-homology recombination (Datsenko
andWanner 2000) as follows. Strains SMR10292 [SMR4562 (F9 DlafU2TFRTcatFRT)] and SMR10308 [SMR4562 (F9 DlafU2T FRTcatFRT dinBo-21(oc))] were used as recipients for deletion by transformation with a DNA fragment amplified from pKD13 with primers yafNwL and yafPwR. Homologous incorporation
of this DNA fragment, which contains the FRTKanFRT marker, results in a deletion of the yafNOP genes. KanR recombinants were selected, and location of the marker


TABLE 2
Plasmids used in this study
Name
Description and source
pFZY
pPdinB
pPdinBOC1
pPdinBOC2


Low-copy plasmid with multicloning site abutting a promoterless lacZ (Koop et al. 1987)
Bases _432 to _2 of dinB from strain SMR4562 cloned into pFZY, producing a PdinBlacZ fusion
Bases _432 to _2 of dinB from strain SMR10308 cloned into pFZY, producing a PdinBo-21(oc)lacZ fusion
Bases _432 to _2 of dinB from strain SMR10309 cloned into pFZY, producing a PdinBo-22(oc)lacZ fusion


in the F9 episome was confirmed both by ability to transfer the resistance during mating and by cotransduction of KanR and CamR (present in the linked lafU2TFRTcatFRT in both strains). The strains resulting from deletion of yafNOP from
the episomes of SMR10292 and SMR10308 were named SMR11023 and SMR11024, respectively. Both strains were
used respectively as P1 donors to transfer the lafU2T FRTcatFRT DyafNOPTFRTKanFRT linkage and the lafU2T
FRTcatFRT dinBo-21(oc) DyafNOP:FRT:KanFRT linkage into the FC231 background, creating strains SMR11026 and SMR11027.
b-Galactosidase assays: b-Galactosidase assays were performed to determine the relative expression of lacZ under
the control of the different versions of the dinB promoter cloned into the low-copy plasmid pFZY (Koop et al. 1987).
Cells were grown in LBH medium until mid-log phase, and the levels of b-galactosidase were determined in samples of the
cultures as described (Miller 1992).
DinB Western blots: For DinB detection on Western blots, stationary-phase cultures grown from single colonies in 5ml of M9 B1 glycerol medium for 48 hr were harvested, and cells were suspended in sample loading/lysis buffer (62.5 mm Tris,
pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 0.5% b-mercaptoethanol), correcting for the OD600 of the
terminal culture. For 1ml of a culture atOD600 of 2 (measured at OD600 #1 with diluted samples), 100 ml of sample loading
buffer was used. Twenty microliters of each sample was separated by electrophoresis on a SDS polyacrylamide gel
(12.5%). Proteins were transferred to a Hybond-LFP PVDF   membrane (Amersham Biosciences), and the membrane was
probed with a polyclonal DinB rabbit antiserum (Kim et al. 2001). A goat anti-rabbit secondary antibody conjugated to the
Cy5 fluorescent dye (Amersham Biosciences) was used for detection of DinB, using the Typhoon scanner (Amersham
Biosciences).
Stress-induced mutagenesis assays: Stress-induced lac reversion assays were performed as described (Harris et al. 1996) with four independent cultures of each strain. The proportion of Lac1 point mutants and lac-amplified colonies
was determined by plating cells from 20 colonies of each culture for each day in which Lac1 colonies were counted
(days 2–5) on LBH rifampicin X-gal plates. This allows the distinction between Lac1 point mutants (solid-blue colonies) and lac-amplified cells, given the lac-unstable sectoring-colony phenotype diagnostic of lac amplification (Hastings et al. 2000).
Determination of the mutation sequences in the lac gene: Lac1 point mutants from experiment day 5 were identified as
described above and purified on LBH plates containing rifampicin and X-Gal. A 300-nucleotide region spanning the lac 11 allele was amplified by PCR using primers lacIL2 (59- AGGCTATTCTGGTGGCCGGA-39 and lacD2 (59-GCCTCTTC
GCTATTACGCCAGCT-39). DNA sequencing was performed by Seqwright (Houston) using primer lacU (59-ATATCCCG
CCGTTAACCACC-39).



Figure 1.—Construction and characterization of two dinB(oc) alleles. (A) Location of the operator-constitutive mutations in the dinB promoter. The SOS operator (from Fernandez De
Henestrosa et al. 2000) is shaded, and the mutations introduced in each of the alleles are in boldface and italic type.
The beginning of the dinB ORF is shown in boldface type. (B) Activity of the dinB promoter in transcriptional fusions with lacZ, measured in both wild-type (SMR4562) and its LexAdefective (null), lexA51(Def), derivative strain SMR5400, in which SOS is constitutively highly induced. The strains from left to right are SMR10838, SMR10839, SMR10840, SMR10841, SMR10842, and SMR10843. PdinB indicates the wild-type dinB promoter present in plasmid pPdinB, POc1 indicates the dinBo- 21(oc) promoter contained in plasmid pPdinBOC1, and POc2 indicates the dinBo-22(oc) promoter contained in plasmid
pPdinBOC2. Mean 6 1 standard error of the mean (SEM) for three independent determinations.



RESULTS

Construction and characterization of the dinB(oc) alleles: To test the hypothesis that dinB might be the sole
SOS gene required at induced levels for stress-induced point mutagenesis, we constructed dinB mutants in which the transcriptional repression by LexA, the repressor controlling the expression of the SOS regulon, is alleviated. This was achieved by site-directed mutagenesis of the dinB promoter, altering the binding site of
the LexA repressor. These are used (see below) to express dinB at SOS-induced levels in strains in which the
rest of the SOS genes are repressed. The sequences of the operator-constitutive dinB(oc) mutations that were constructed are shown in Figure 1A.
To test whether these mutations behave as bona fide operator-constitutive alleles, we fused the dinB promoter regions from the two dinB(oc) alleles to lacZ and measured
the levels of b-galactosidase expression from these PdinBlacZ fusions carried in a low-copy plasmid (Figure
1B). Introduction of these plasmids into wild-type cells resulted in _10-fold higher lacZ expression from both  PdinB(o ) c lacZ fusions when compared with wild-type PdinB.nThis is in agreement with previous estimates of transcriptional induction of dinB during the SOS response (Courcelle et al. 2001; Kim et al. 2001). lexA51(Def)
cells have no functional LexA repressor and show constitutive SOS expression (Mount 1977). We find that lacZ expression is increased in a lexA51(Def) strain when driven by the wild-type dinB promoter, but see no significant increase with the dinB(oc) promoters, showing that levels of dinB transcription similar to that achieved by true SOS derepression are achieved by the dinB(oc) mutations. The lexA51(Def) strain SMR5400 also carries a mutation in the sulA gene, which allows survival under constitutive SOS induction (Mount 1977), and a mutation in the F-encoded psiB gene, which has been shown to exert a negative effect on stressinducedmutagenesis (McKenzie et al. 2000) probably by affecting SOS induction (reviewed by Cox 2007).
In the Lac-assay strains such as FC40 and SMR4562, dinB is present both in the chromosome and in the F9128, at which locus it is more highly expressed (Kim et al. 2001). Introduction of both dinB(oc) alleles into
the episomal dinB locus results in about five- to six-fold increased DinB-protein levels in stationary-phase cells
compared with an otherwise isogenic SMR4562 derivative in both wild-type and lexA3(Ind_) backgrounds
(Figure 2). This indicates that both dinB(oc) alleles are functional in vivo, conferring an increased basal dinB
expression. Furthermore, both alleles confer levels of expression similar to those observed in lexA51(Def) cells
(Figure 2), at least in the growth conditions used by us in the stress-induced mutagenesis experiments (cells
grown for 48 hr in M9 B1 glycerol minimal medium). It was noted before (Kim et al. 2001) that expression of
dinB in the F9128 plasmid is higher than that from the chromosomal dinB. Our finding that both dinB(oc)
alleles, when present only in the episome, increase DinB to levels similar to that observed in the lexA51(Def)
strain (in which both the episomal and the chromosomal copy are constitutively highly expressed), also implies that the episomal expression is more pronounced than the chromosomal expression. To facilitate further strain construction and genetic analysis, we carried out the subsequent experiments in cells bearing a single dinB(oc) allele in the F9128 plasmid.






Figure 2.—DinB Western blots. Stationary-phase cells grown in M9 B1 glycerol medium were harvested and analyzed
using a rabbit polyclonal DinB antiserum as described (materials and methods). Values shown represent the average
DinB protein levels relative to wild type determined in three independent experiments 6 SEM. Similar results were
obtained with Western blots performed with a DinB monoclonal antibody. Strains are the following: dinB, SMR5889; dinB1,
SMR10292; dinBo-21(oc), SMR10308; dinBo-22(oc), SMR10309; lexA3(Ind_), SMR10760; lexA3(Ind_) dinBo- 21(oc), SMR10761; lexA3(Ind_) dinBo-22(oc), SMR10762; and lexA(Def), SMR5400.


dinB(oc) mutations restore stress-induced point mutagenesis in SOS off strains: Because DinB is a key player in stress-induced mutagenesis, we wanted to examine whether dinB is the only gene required at SOS-induced levels for stress-induced point mutagenesis in the Lac assay. The SOS response is induced when DNA damage is sensed in the form of single-strand DNA (reviewed by Friedberg et al. 2005). RecA binds the single-strand DNA and becomes active as a co-protease that facilitates cleavage of the LexA repressor, resulting in upregulation of the SOS genes, including dinB. To determine whether dinB upregulation constitutes the sole role of the SOS response in stress-induced point mutagenesis, we tested the effect of the dinB(oc) alleles on lac reversion in both wild-type and lexA3(Ind_) backgrounds. The lexA3(Ind_) mutation creates an uncleavable LexA/SOS repressor such that derepression of the SOS response genes during an SOS response is prevented (Mount et al. 1972). Previously, this allele was shown to cause reduced stress-induced point mutagenesis in the Lac assay (Cairns and Foster 1991; McKenzie et al. 2000, 2001), indicating that one or more SOS-controlled genes are needed at induced levels for efficient stress-induced mutagenesis. Representative results from single experiments with each of the two
dinB(oc) alleles constructed are shown in Figure 3, A and B, and quantification of the stress-induced point
mutagenesis rates from multiple experiments is shown in Figure 3C. Strikingly, either allele provides a complete
suppression of the phenotype of the lexA3(Ind_) strain. These results show that the reduced stressinduced
mutagenesis in a lexA3(Ind_) strain is caused specifically by the failure to upregulate dinB, and not
any other gene in the LexA/SOS regulon. This finding places DinB as the central SOS-regulated protein in
stress-induced mutagenesis and indicates that upregulation of other SOS genes such as recA, ruvA, and ruvB beyond their constitutive levels of expression is irrelevant.



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