reca-sos

8/2/2019 RecA-SOS

1/12

JOURNAL OF BACTERIOLOGY, Nov. 2005, p. 76077618 Vol. 187, No. 220021-9193/05/$08.000 doi:10.1128/JB.187.22.76077618.2005Copyright 2005, American Society for Microbiology. All Rights Reserved.

Roles of the Escherichia coli RecA Protein and the Global SOSResponse in Effecting DNA Polymerase Selection In Vivo

Robert W. Maul and Mark D. Sutton*

Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University ofNew York, Buffalo, New York 14214

Received 8 July 2005/Accepted 25 August 2005

The Escherichia coli sliding clamp protein is proposed to play an important role in effecting switchesbetween different DNA polymerases during replication, repair, and translesion DNA synthesis. We recentlydescribed how strains bearing the dnaN159 allele, which encodes a mutant form of the clamp (159), displaya UV-sensitive phenotype that is suppressed by inactivation of DNA polymerase IV (M. D. Sutton, J. Bacteriol.186:67386748, 2004). As part of an ongoing effort to understand mechanisms of DNA polymerase managementin E. coli, we have further characterized effects of the dnaN159 allele on polymerase usage. Three of the five

E. coli DNA polymerases (II, IV, and V) are regulated as part of the global SOS response. Our results indicatethat elevated expression of the dinB-encoded polymerase IV is sufficient to result in conditional lethality of the

dnaN159 strain. In contrast, chronically activated RecA protein, expressed from the recA730 allele, is lethal to

the dnaN159 strain, and this lethality is suppressed by mutations that either mitigate RecA730 activity (i.e.,recR), or impair the activities of DNA polymerase II or DNA polymerase V (i.e., polB or umuDC). Thus,

we have identified distinct genetic requirements whereby each of the three different SOS-regulated DNApolymerases are able to confer lethality upon the dnaN159 strain, suggesting the presence of multiple mech-anisms by which the actions of the cells different DNA polymerases are managed in vivo.

The finding that Escherichia coli possesses five distinctDNA polymerases, while humans are currently known topossess at least three times this many (reviewed in refer-ences 15 and 61), suggests that organisms must employ aseries of adaptable control systems in order to ensure thatthe correct DNA polymerase gains access to the replicationfork at the appropriate time. To some extent, a cell can

regulate the access to the replication fork of its differentDNA polymerases by attenuating their respective expressionlevels, specific activities, and/or subcellular localizations (reviewed in reference 61). However, even with these controlsystems in place, situations will inevitably arise in which acell must choose between multiple DNA polymerases. Asselection of the incorrect polymerase could have cata-strophic consequences, it seems likely that control systemsin addition to those mentioned above must exist in order toensure that in these situations, the correct DNA polymeraseis recruited to the replication fork at the appropriate time.

When considering how E. coli might manage the actions ofits five DNA polymerases to coordinately regulate DNA rep-lication with DNA repair and translesion DNA synthesis, onecannot overstate the importance of the global SOS response:the steady-state levels of polymerase II (Pol II), Pol IV, andPol V vary as much as 10-fold or more as a function of SOSinduction (23). Proper management of the E. coli SOS re-sponse requires the products of the lexA and recA genes.LexA protein acts as a transcriptional repressor. By binding tosequences termed SOS boxes located near promoters, LexA

blocks access of RNA polymerase to the promoter, therebyeffectively repressing transcription of more than 40 differentgenes (10, 16).

RecA protein, which is required for most homologous re-combination (reviewed in reference 30), as well as for therepair of double-stranded DNA breaks (reviewed in references24 and 30) and the restart of stalled replication forks (reviewed

in reference 11), becomes activated for its role in SOS induc-tion by binding to single-stranded DNA (ssDNA), which pre-sumably results from the cells failed attempts to copy overlesions in the DNA (reviewed in references 16 and 60). Uponbinding to ssDNA, RecA forms a helical filament structure,referred to as activated RecA (30). Interaction of LexA withactivated RecA mediates autodigestion of LexA, which largelyinactivates its repressor function, leading to transcriptionalderepression of the LexA-regulated genes (reviewed in refer-ence 16).

Although many of the SOS-regulated genes, including polB(Pol II) and dinB (Pol IV), are expressed at modest levels inthe absence of DNA damage, others, such as umuDC (Pol V),are expressed at easily detectable levels only following SOSinduction (16, 60). Moreover, catalytic activity of Pol V re-quires that its umuD-encoded subunit undergo a RecA/ssDNA-facilitated autodigestion that is mechanistically similarto that of LexA (7, 41, 45, 53): intact UmuD, together withUmuC, acts in a DNA damage checkpoint control (38, 43), while autodigestion of UmuD results in the removal of itsN-terminal 24 residues to generate UmuD (7, 41, 53), whichleads to the release of the checkpoint and concomitant activa-tion of Pol V-dependent translesion DNA synthesis. In addi-tion to its role in the expression and posttranscriptional acti-vation of Pol V, RecA also plays an as yet poorly understoodrole in stimulating the catalytic activity of Pol V (46, 47, 49, 52,61).

* Corresponding author. Mailing address: Department of Biochemis-try, 140 Farber Hall, School of Medicine and Biomedical Sciences, StateUniversity of New York at Buffalo, 3435 Main Street, Buffalo, NY 14214.Phone: (716) 829-3581. Fax: (716) 829-2661. E-mail: [email protected].

7607

8/2/2019 RecA-SOS

2/12

In addition to SOS regulation, work from a variety of labo-ratories (2, 5, 8, 12, 27, 29, 67), including our own (5659),indicates that the E. coli sliding clamp and the clamp loadercomplex play important roles in coordinating access of thedifferent DNA polymerases to the replication fork. The clamp is a ring-shaped, homodimeric protein that topologicallyencircles double-stranded DNA. It is loaded onto DNA by the complex, which is comprised of six distinct polypeptides (2,, , , , and ) (6, 34). Although the mechanism by whichthe clamp is loaded onto DNA is not yet fully understood, itis clear that complex binds to the clamp in an ATP-dependent fashion to catalyze the opening of the clamp. Afterpositioning the opened clamp on the DNA so that it encirclesdouble-stranded DNA, the complex undergoes a conforma-tional change that is triggered by the ATPase activity of the and subunits, which effects release of the clamp, resultingin its loading (1, 20). Once loaded, the clamp moves freelyalong the DNA, acting as a mobile platform (55).

All five E. coli DNA polymerases interact with , and thisinteraction serves to tether the polymerases to the DNA, en-

dowing them with various degrees of processivity (5, 29, 31, 33,63, 67). In the case of Pol IV, this interaction also serves toenhance its affinity for deoxyribonucleotides (3). Moreover, ithas recently become clear that all five DNA polymerases bindto overlapping surfaces on the clamp (28, 69), suggesting thatcompetition of the different DNA polymerases for interactionwith the clamp may help to coordinate polymerase access tothe replication fork (12, 28, 66, 67, 69).

Consistent with the idea that the clamp and the clamploader complex help to coordinate access to the replication forkof the different DNA polymerases, Viguera et al. (65) determinedthat a holD::kan mutation, which affects the subunit of the clamp loader complex, conferred a temperature-sensitive phe-

notype that was suppressed by deletion of polB, and to a lesserextent by deletion of dinB. Furthermore, we previously reportedthat the dnaN159 allele, which encodes a mutant form of the sliding clamp bearing G66E and G174A substitutions (19, 42, 56),was impaired for interaction with the catalytic subunit of Pol IIIand conferred a UV-sensitive phenotype in a nucleotide excisionrepair-deficient background that was suppressed by (not epistaticwith) inactivation of Pol IV (56). Taken together, these findingssuggest that mutations that impair either the clamp or theefficiency of clamp loading lead to altered polymerase usage,consistent with a role for the clamp in helping to regulate Polaccess to the replication fork.

Presumably, the ability of the SOS-regulated DNA poly-merases to impair growth of a dnaN159 or holD::kan strain isdue in large part to a replication defect conferred by themutations. Although it is possible that the various polymerasescompete with each other for access to the replication fork in astochastic manner, the fact that only particular SOS-regulatedDNA polymerases impaired growth in specific genetic back-grounds suggested to us that additional factors, such as thesteady-state levels of the different DNA polymerases, whichare heavily influenced by the SOS response, as well as otherSOS-regulated gene products, could help regulate access of theSOS-regulated polymerases to the replication fork.

To test this hypothesis, we used different lexA and recAalleles to determine the individual effects of SOS induction andRecA activation on polymerase usage in the dnaN159 mutant.

Our results indicate that elevated levels of Pol IV impairedgrowth of the dnaN159 mutant. However, the recA730 allele,which expresses a mutant form of the RecA protein (RecA730)that displays an enhanced ability to form RecA/ssDNA nucleo-protein filaments, conferred a Pol II- and Pol V-dependentlethality in the dnaN159 mutant. Lethality conferred by Pol Vdid not correlate with its expression level, but rather was de-pendent upon recA730. Taken together, these results suggestthat the LexA and RecA proteins play an important role ineffecting polymerase selection and/or polymerase switching atthe replication fork following DNA damage. These findings arediscussed in terms of a model to describe how E. coli managesthe actions of its different DNA polymerases.

MATERIALS AND METHODS

E. coli strains and bacteriological techniques. E. coli strains are described in

Table 1 and were routinely grown in Luria-Bertani (LB) medium (36). Whennecessary, the following antibiotics were used at the indicated concentrations:ampicillin, 150 g/ml; chloramphenicol, 20 g/ml; kanamycin, 60 g/ml; specti-

nomycin, 50 g/ml; streptomycin, 50 g/ml; tetracycline, 10 g/ml; and rifampin,

100 g/ml. All strains are derivatives of E. coli K-12 and were constructed usingP1vir-mediated generalized transduction (36). Transduction of the recA730 and

recA441 alleles was achieved by selection for tetracycline resistance conferred bythe closely linked srlC300::Tn10 allele, and the correct sequence for each allele

was confirmed by automated nucleotide sequence analysis of the PCR-amplifiedrecA gene. Transduction of the dnaN159 allele was achieved by selection for theclosely linked tnaA300::Tn10 or zid-3162::Tn10kan alleles and was confirmed by

verifying temperature sensitivity and by nucleotide sequence analysis of thePCR-amplified dnaN allele, as previously described (56).

Transduction of the (araD-polB)::, (dinB-yafN)::kan, and umuDC595::catalleles was done by virtue of antibiotic resistance conferred by the indicated marker.Due to the high background of spectinomycin-resistant CFU occasionally observed

when selecting for strains bearing the fragment (regardless of the spectinomycinconcentration used; data not shown), we transduced strains that we intended tomake (araD-polB):: to tetracycline resistance with leu::Tn10, which is adjacent to

polB, and then replaced leu::Tn10 with (araD-polB)::. The presence of each allele

was subsequently confirmed by colony PCR. The

(araD-polB

)::

allele was con-firmed using primers 5-CCG ACG GGA TCA ATC AGA AAG GTG-3 and5-TCT GTC CTG GCT GGG AAC GA-3, which amplify a 817-base-pair frag-ment from (araD-polB):: and yield no product using the polB allele.

The (dinB-yafN)::kan allele was confirmed using primers 5-cgc gaa ttc catATG CGT AAA ATC ATT CAT GTG GAT ATG G-3 (the first 12 bases bear

no sequence similarity to the dinB gene; rather, they introduce an NdeI restric-tion site that was used for cloning dinB into an overexpression vector) and5-CCG GTT GAT CAA TAA AGT ATT TAG CTG GG-3 , which amplify a1,000-base-pair fragment from the dinB-yafN region, and a 1,250-base-pair fragment from (dinB-yafN)::kan. The umuDC595::cat allele was con-firmed using primers 5-AGG CCA CGT GAG CAC AAG ATA AGA-3 and

5-ATA GGT ACA TTG AGC AAC TGA CTG-3, which amplify a 530-base-pair fragment from umuDC595::cat strains and yield no product using the

umuDC alleles.Plasmid construction and transformation assay. Plasmid DNAs are described

in Table 1. Genes encoding Pol I (polA), Pol II (polB), and Pol IV (dinB) were

PCR amplified from genomic DNA, and a synthetic operon encoding Pol V(umuDC) was PCR amplified from plasmid pGW3751 (41) using the following

primer pairs: polA-promoter, 5-CTT GCG TGA AAC GGG CGC CTT-3 andpolA-end, 5-ggg aca cct agg TTA GTG CGC CTG ATC CCA G-3 (the first 12nucleotides are not complementary to polA and were included for cloning pur-

poses); polB-promoter, 5-CAC TAT CTG CGT AAG CAT GGC GCG AAGGC-3 and polB-end, 5-ggg aca cct agg TCA GAA TAG CCC AAG TTG C-3

(the first 12 nucleotides are not complementary to polB and were included for

cloning purposes); dinB-promoter, 5-CAA TAA GAA TTC CGT CAA TCGCCA TCT GTT TGC CGG G-3 and dinB-end, 5-cgc aca aag ctt ggt acc TCA

TAA TCC CAG CAC CAG TTG TC-3 (the first 18 nucleotides are not com-plementary to dinB); and umuDC-promoter, 5-CTG CTG GCA AGA ACAGAC-3 and umuDC-end, 5-CGT GAT CTG TTC GGT CGC TAA TCC-3.

PCR fragments were blunt-end ligated into pCR-BluntII-Topo vector (Invitro-gen, Carlsbad, CA) as per the manufacturers recommendations. After verifying

that each clone contained the correct nucleotide sequence (Roswell Park Cancer

7608 MAUL AND SUTTON J. BACTERIOL.

8/2/2019 RecA-SOS

3/12

Institute Biopolymer Facility, Buffalo, NY), fragments were subcloned into pWSK29by digestion with EcoRI. The correct orientation (downstream of the lac promoter)

was confirmed by diagnostic PCR using the M13 reverse primer, which is homolo-gous to the sequence upstream of the multiple cloning site in pWSK29, paired with

second primer homologous to the 3-end of the cloned insert.

Transformation assays were preformed with the indicated E. coli strains whichwere made chemically component using rubidium chloride (51). Fifty microliters

of component cells (5.2 108 cells) was incubated with 200 ng of each purifiedplasmid DNA on ice for 30 min. Reactions were heat shocked at 42C for 2 min,

followed by incubation at 30C for 1 h. Aliquots of each reaction were then

TABLE 1. E. coli strains and plasmids used in this studya

Strain or plasmid Relevant genotype Source or construction

StrainAM107 recR252::Tn109kan J. Courcelle (9)CAG18497 fadR613::Tn10 CGSCb

CAG18558 zid-3162::Tn10kan B. Michel (19)DE298 recA441 srlC300::Tn10 R. Woodgate

FC1237

(dinB-yafN

)::kan

P. Foster (54)GW8028 ara-714 leu::Tn10 G. WalkerJJC1209 dnaN159 zid-3162::Tn10 B. Michel (19)RW120 umuDC595::cat R. Woodgate (71)STL1336 (araD-polB):: M. Goodman (4)MS100 tnaA300::Tn10 This laboratory (56)MS101b dnaN159 tnaA300::Tn10 This laboratory (56)RW542 lexA51(Def) R. Woodgate (21)MS104 lexA51(Def) tnaA300::Tn10 This laboratory (56)MS105 lexA51(Def) dnaN159 tnaA300::Tn10 This laboratory (56)RM100 lexA51(Def) (dinB-yafN)::kan P1(FC1237) RW542RM101 lexA51(Def) umuDC595::cat P1(RW120) RW542RM102 lexA51(Def) ara-714 leu::Tn10 P1(GW8028) RW542RM103 lexA51(Def) (araD-polB):: P1(STL1336) RM102RM104 lexA51(Def) (araD-polB):: tnaA300::Tn10 P1(MS100) RM103RM105 lexA51(Def) (araD-polB):: dnaN159 tnaA300::Tn10 P1(MS101) RM103RM106 lexA51(Def) (dinB-yafN):: kan tnaA300::Tn10 P1(MS100) RM100RM107 lexA51(Def) (dinB-yafN):: kan dnaN159 tnaA300::Tn10 P1(MS101) RM100RM108 lexA51(Def) umuDC595:: cat tnaA300::Tn10 P1(MS100) RM101

RM109 lexA51(Def) umuDC595:: cat dnaN159 tnaA300::Tn10 P1(MS101) RM101RW540 lexA51(Def) uvrA6 R. WoodgateRM110 lexA51(Def) uvrA6 tnaA300::Tn10 P1(MS100) RW540RM111 lexA51(Def) uvrA6 dnaN159 tnaA300::Tn10 P1(MS101) RW540RM112 lexA51(Def) uvrA6 recA441 srlC300::Tn10 P1(DE298) RW576RM113 lexA51(Def) uvrA6 recA441 srlC300::Tn10 zid3162::Tn10kan P1(CAG18558) RM112RM114 lexA51(Def) uvrA6 recA441 srlC300::Tn10 dnaN159 zid-3162::Tn10kan P1(JJC1209) RM112RW576 lexA51(Def) uvrA6 recA730 R. WoodgateRM115 lexA51(Def) uvrA6 recA730 tnaA300::Tn10 P1(MS100) RW576RM116 lexA51(Def) uvrA6 recA730 (dinB-yafN)::kan P1(FC1237) RW576RM117 lexA51(Def) uvrA6 recA730 (dinB-yafN):: kan tnaA300::Tn10 P1(MS100) RM116RM118 lexA51(Def) uvrA6 recA730 umuDC595::cat P1(RW120) RW576RM119 lexA51(Def) uvrA6 recA730 umuDC595:: cat tnaA300::Tn10 P1(MS100) RM118RM120 lexA51(Def) uvrA6 recA730 umuDC595:: cat dnaN159 tnaA300::Tn10 P1(MS101) RM118RM121 lexA51(Def) uvrA6 recA730 ara-714 leu::Tn10 P1(GW8028) RW576RM122 lexA51(Def) uvrA6 recA730 (araD-polB):: P1(STL1336) RM121RM123 lexA51(Def) uvrA6 recA730 (araD-polB):: tnaA300::Tn10 P1(MS100) RM122RM124 lexA51(Def) uvrA6 recA730 (araD-polB):: dnaN159 tnaA300::Tn10 P1(MS101) RM122

RM125 umuDC595:: cat fadR613::Tn10 P1(CAG18497) RW120RM126 lexA51(Def) uvrA6 recA730 (araD-polB):: umuDC595:: cat fadR613::Tn10 P1(RM125) RM122RM127 lexA51(Def) uvrA6 recA730 (araD-polB):: umuDC595:: cat fadR613::Tn10 zid-3162::Tn10kan P1(CAG18558) RM126RM128 lexA51(Def) uvrA6 recA730 (araD-polB):: umuDC595:: cat fadR613::Tn10 dnaN159 zid3162::Tn10kan P1(JJC1209) RM126RM129c lexA51(Def) uvrA6 recA730 dnaN159 tnaA300::Tn10 srd-1 P1(MS101) RW576RM130c lexA51(Def) uvrA6 recA730 dnaN159 tnaA300::Tn10 srd-2 P1(MS101) RW576RM131a lexA51(Def) uvrA6 recA730 dnaN159 tnaA300::Tn10 srd-1 umuDC595::cat P1(RW120) RM129RM132 lexA51(Def) uvrA6 recA730 recR252::Tn109kan P1(AM107) RW576RM133 lexA51(Def) uvrA6 recA730 recR252::Tn109kan tnaA300::Tn10 P1(MS100) RM132RM134 lexA51(Def) uvrA6 recA730 recR252::Tn109kan dnaN159 tnaA300::Tn10 P1(MS101) RM132RM135c lexA51(Def) uvrA6 recA730 zid-3162::Tn10kan srd-1 P1(CAG18558) RM129RM136c lexA51(Def) uvrA6 recA730 zid-3162::Tn10kan srd-2 P1(CAG18558) RM130

PlasmidspWSK29 Apr; pSC101 derivative S. Kushner (68)pRM100 Apr; pWSK29 bearing polA (Pol I) This workpRM101 Apr; pWSK29 bearing polB (Pol II) This workpRM102 Apr; pWSK29 bearing dinB (Pol IV) This work

pRM103 Apr

; pWSK29 bearing umuD and umuC (Pol V) This worka See Materials and Methods for a description of specific strain constructions. Strains AM107 to STL1336 (lines 1 to 9 in the table) were used as donors for

P1vir-mediated transduction. Strains MS100 to RM109 (lines 10 to 24 in the table) and RM125 (line 42) are derivatives of strain RW118 [thr-1 araD139 (gpt-proA)62lacY1 tsx-33 glnV44(AS) galK2(Oc) hisG4(Oc) rpsL31 xylA5 mtl-1 argE3(Oc) thi-1 sulA211]. Strains RM110 to RM136 (lines 26 to 53 in the table), except for RM125,are derivatives of strain RW540 [line 25 in the table; thr-1 araD139 (gpt-proA)62 lacY1 tsx-33 glnV44(AS) galK2(Oc) hisG4(Oc) rpsL31 xylA5 mtl-1 argE3(Oc) thi-1 sulA211 leuB6(Am) uvrA6 recA730].

b The dnaN159 allele, which encodes two amino acid substitutions (G66E and G174A), was originally referred to as dnaN59 but has since been renamed by the E. coliGenetic Stock Center (CGSC).

c These strains contain an unknown mutation that suppresses recA730 dnaN159 synthetic lethality. These suppressor loci are referred to as srd-1 and srd-2, forsuppressor of recA730 dnaN159 synthetic lethality. See the footnotes to Table 3 and the text for further details.

VOL. 187, 2005 RecA AND DNA POLYMERASE SELECTION 7609

8/2/2019 RecA-SOS

4/12

plated onto LB plates supplemented with ampicillin, and colonies were countedafter overnight incubation at 30C.

Mutagenesis assays. UV-induced mutagenesis was preformed using culturesgrown overnight in M9 minimal medium supplemented with glucose (0.2%),

thiamine (1 g/ml), and Casamino Acids (0.5%) essentially as described previ-ously (56, 57). Briefly, overnight cultures were subcultured in the same medium

at 30C with shaking until they reached mid-exponential growth (optical densityat 595 nm [OD595] of 0.6). One milliliter of culture was then transferred to asterile glass 15 mm petri dish and either irradiated with 3 J/m2 UV light (254 nm)

using a 15-watt germicidal bulb (General Electric) or mock-irradiated. Cultureswere then transferred to sterile 25 mm glass bubbler tubes containing 9 ml of

supplemented M9 medium, followed by overnight incubation at 30C with shak-ing. The following day, 100-l aliquots of the irradiated and mock-irradiatedovernight cultures were plated in duplicate onto LB plates containing rifampin in

order to measure mutagenesis, while appropriate dilutions were plated onto LBwithout rifampin in order to determine cell titers. UV-induced mutation fre-

quency was then calculated by determining the number of rifampin-resistant(Rifr) colonies following UV irradiation minus the number of Rifr coloniesinduced by mock UV irradiation divided by the cell titer. Spontaneous mutagen-

esis was measured in a similar manner except that cultures were not UV irradi-ated.

RESULTS

Access to the replication fork of the dinB-encoded Pol IV is

regulated in part by its steady-state level. We previously re-ported that the lexA51(Def) allele exacerbated the tempera-ture-sensitive growth phenotype of the dnaN159 strain suchthat it was unable to grow at temperatures above 34C (56).Since (i) the UV sensitivity of the dnaN159 strain was sup-pressed by inactivation of Pol IV and (ii) the dinB-encoded PolIV is SOS regulated, we hypothesized that the enhanced tem-perature sensitivity of the dnaN159 lexA51(Def) strain wasattributable to elevated levels of Pol IV. We tested this hy-pothesis in two different ways. In our first experiment, weasked whether SOS-induced levels of Pol IV or one of theother SOS-regulated DNA polymerases (i.e., Pol II or Pol V)led to the growth defect of the dnaN159 lexA51(Def) strain.For this, we compared the growth properties of various iso-genic lexA51(Def) strains bearing either dnaN or dnaN159together with deletions of polB (Pol II), dinB (Pol IV), orumuDC (Pol V). Our results indicated that inactivation of PolIV [(dinB-yafN)::kan] suppressed the temperature-sensitivegrowth phenotype of the dnaN159 lexA51(Def) strain at both

35 and 37C (Table 2). In contrast, inactivation of Pol II[(araD-polB)::] o r Po l V (umuDC595::cat) had only aminimal effect on the growth phenotype of the dnaN159lexA51(Def) strain.

In our second experiment, we asked whether plasmids ex-pressing DNA polymerase I, II, IV, or V impaired growth ofthe dnaN lexA(Def) and/or the dnaN159 lexA51(Def) strainsdescribed above. As shown in Fig. 1A, each of these plasmidstransformed strain MS104 [dnaN lexA51(Def)] with an effi-ciency that was comparable to that of the pWSK29 controlplasmid. In contrast, we were unable to obtain more than a fewtransformants of the dnaN159 lexA51(Def) strain (MS105) us-ing the Pol IV-expressing plasmid. With the exception of the

Pol I-expressing plasmid, which displayed a 10-fold reductionin transformation efficiency, the other plasmids behaved likethe pWSK29 control plasmid with respect to their transforma-tion efficiency (Fig. 1A). Addition of isopropylthiogalactopyr-anoside (IPTG) to 50 M, which leads to maximal induction ofthe lac promoter in pWSK29 (57) (data not shown), had noeffect on these results (data not shown). Very similar results were observed using strains RM110 [dnaN lexA51(Def)uvrA6] and RM111 [ dnaN159 lexA51(Def) uvrA6] (Fig. 1B),indicating that, unlike the UV sensitivity of the dnaN159strain that was severely enhanced by the uvrB::cat or uvrC::cat allele (56), the Pol IV-dependent growth defect of thednaN159 strain was independent of nucleotide excision repair.Taken together, these results suggest that LexA-dependentSOS regulation of the Pol IV steady-state level is important forhelping to regulate its access to the replication fork.

Synthetic lethality of the dnaN159 recA730 strain is sup-

pressed by mutations that inactivate DNA Pol II or DNA Pol

V. Results discussed above suggest that access of Pol IV to thereplication fork can be regulated, at least in part, by its steady-state level relative to the other polymerases. RecA protein isrequired for induction of the global SOS response, as well asmost homologous recombination, the repair of double-stranded DNA breaks, the restart of stalled replication forks,and Pol V-dependent translesion DNA synthesis (reviewed inreferences 11, 16, 24, 30, and 61). In order to determinewhether RecA affects polymerase selection following SOS in-

TABLE 2. Plating efficiencies of different isogenic lexA51(Def) strains

StrainaRelevant genotypeb Efficiency of growthc (relative to growth at 30C):

dnaN lexA polB dinB umuDC 35C 37C 39C

MS100 1.1 1.1 1.0MS101 159 1.4 1.1 1.9 104

MS104 51(Def) 1.0 1.4 0.41

MS105 159 51(Def) 3.0 103

2.0 103

1.0 106

RM104 51(Def) 1.2 1.4 0.75RM105 159 51(Def) 1.0 103 2.0 104 1.0 106

RM106 51(Def) 4.3 3.9 3.2RM107 159 51(Def) 2.6 1.9 1.0 106

RM108 51(Def) 2.1 1.1 2.9RM109 159 51(Def) 4.0 102 8.1 102 1.0 106

a Strains are described in Table 1.bAbbreviations: , wild type; 159, dnaN159; 51(Def), lexA51(Def); , deletion; polB (Pol II), (araD-polB)::; dinB (Pol IV), (dinB-yafN)::kan; and umuDC

(Pol V), umuDC595::cat.c Representative clones for each strain were grown overnight in LB medium at 30C, and serial dilutions of each culture were plated at 30, 35, 37, and 39C. Values

shown are the ratio of CFU observed at the indicated temperature after overnight incubation divided by the number observed for the same strain at 30C. The numberof CFU/ml observed at 30C for each strain was: MS100, 5.3 108; MS101, 3.8 108; MS104, 1.9 108; MS105, 2.1 108; RM104, 2.4 108; RM105, 3.4 108;RM106, 0.73 108; RM107, 0.54 108; RM108, 0.65 108; and RM109, 0.59 108.


8/2/2019 RecA-SOS

5/12

duction, we combined dnaN159 with different recA alleles un-der the premise that certain combinations might confer syn-thetic phenotypes that could be modified by mutationsaffecting one or more of the various E. coli polymerases. In-activation of recA (recA::cat) had only a minor effect on thednaN159 strain, resulting in a slightly enhanced temperature-sensitive growth phenotype (data not shown), consistent withprevious reports (19). This phenotype was similar to that of the dnaN159 lexA3(Ind) strain (56), suggesting that it resultedfrom a lack of Pol V, and was not further characterized.

We next examined recA alleles that displayed enhancedDNA binding activity. The recA441 allele, which bears E38Kand I298V substitutions, displays an enhanced rate of associ-ation with ssDNA in vitro (25, 26). The recA730 allele, whichwas isolated from recA441 by Witkin et al. (70), contains onlythe E38K substitution, and the mutant RecA730 protein dis-plays an even more robust ssDNA binding activity in vitro thandoes RecA441 (25). Despite the fact that we could efficientlytransduce the dnaN159 allele into the recA441 lexA51(Def)strain (RM112) by selection for kanamycin resistance con-

ferred by the nearby zid-3162::Tn10kan allele (Table 3), trans-duction ofdnaN159 into the isogenic recA730 lexA(Def) strainRW576 was remarkably inefficient (Table 3). Similar resultswere observed when trying to transduce dnaN159 into anotherrecA730 strain [NR9350 (genotype: ara thi (pro-lac) sulA211srlC300::Tn10 recA730) (13)] using a P1vir lysate prepared ona zid-3162::Tn10kan dnaN159 strain (data not shown), indicat-ing that the inability to do so was specific to combination ofdnaN159 and recA730 and not to some other allele(s).

In order to rule out the possibility that the recA730 mutationsuppressed the temperature-sensitive growth phenotype of thednaN159 strain, we determined the nucleotide sequence of thePCR-amplified dnaN allele from six separate strains; all six

corresponded to the wild-type dnaN sequence (data notshown). This result indicates that recA730 does not suppressthe temperature-sensitive growth phenotype of dnaN159 andsuggests that the combination of the dnaN159 and recA730alleles results in a synthetic lethal phenotype.

Given our finding that SOS-induced levels of Pol IV exac-erbated the temperature-sensitive growth phenotype of thednaN159 mutant (Table 2), we hypothesized that SOS-inducedlevels of one or more of the three SOS-regulated DNA poly-merases, in combination with RecA730, might result in lethal-ity in the dnaN159 strain. We therefore constructed a series ofisogenic RW576 derivatives in which we combined the recA730allele with mutations inactivating each of the SOS-regulatedpolymerases (Pol II, Pol IV, and Pol V) and tested these strainsfor their ability to be transduced to temperature sensitivity withdnaN159.

In striking contrast to our results discussed above indicatingthat inactivation of Pol IV suppressed the growth defect of thednaN159 lexA51(Def) strain (see Table 2 and Fig. 1), inactiva-tion of Pol IV [(dinB-yafN)::kan] had no discernible effecton the transduction efficiency of dnaN159 into the recA730strain (Table 3). Conversely, inactivation of either Pol II[(araD-polB)::] or Pol V (umuDC595::cat) allowed effi-cient transduction of dnaN159 into the recA730 strain (Table3). Furthermore, inactivation of both polB and umuDC in thesame strain (RM126) resulted in an even slightly higher effi-ciency of transduction for dnaN159 (Table 3). These findings

FIG. 1. Effects of plasmids directing expression of DNA polymer-ase I, II, IV, or V on growth of different dnaN and dnaN159

lexA51(Def) strains. Plasmids expressing Pol I, II, IV, or V were trans-formed into E. coli strains MS104 [dnaN lexA51(Def)] and MS105[ dnaN159 lexA51(Def)] (A) and RM110 [dnaN lexA51(Def) uvrA6]and RM111 [dnaN159 lexA51(Def) uvrA6] (B). Transformation effi-ciencies are expressed as the number of CFU obtained per microgramof supercoiled plasmid DNA (note that the y-axis is a log10 scale).Values represent the averages and ranges of two independent exper-iments. Plasmids: pWSK29, control; pRM100, polA (Pol I); pRM101,

polB (Pol II); pRM102, dinB (Pol IV); pRM103, umuDC (Pol V).


8/2/2019 RecA-SOS

6/12

indicate that lethality of the dnaN159 recA730 strain is theresult of Pol II and/or Pol V. The fact that UmuD levels were

similar in the recA441 and recA730 strains (Fig. 2) indicatesthat lethality is not due simply to the presence of elevatedlevels of Pol V or to the checkpoint function ofumuDC (sinceintact UmuD was poorly detectable), but rather some propertyof the RecA730 protein in the dnaN159 strain which affectsone or more functions of Pol II and Pol V.

Genetic characterization of viable dnaN159 recA730 strains

provides further evidence for a role of DNA polymerase V-

dependent replication in conferring synthetic lethality. De-spite the fact that the dnaN159 allele was synthetically lethalwith recA730, we were able to identify two dnaN159 transduc-tants of the recA730 strain that acquired temperature-sensitivegrowth (RM129 and RM130; see Tables 1 and 3). Based onnucleotide sequence analysis, these two strains did in fact con-tain the recA730 and dnaN159 alleles (data not shown). Inaddition, both strains contained the correct sequence for polB(Pol II) and umuDC (Pol V) (data not shown). In order todetermine whether these strains contained a suppressor of thesynthetic lethality, we first replaced the dnaN159 allele in RM129and RM130 with dnaN by selection for the nearby zid-3162::Tn10kan allele (which is located on the opposite side ofdnaN,as is tnaA300::Tn10), resulting in strains RM135 and RM136,respectively (see Table 3 footnotes). We next attempted totransduce strains RM135 and RM136 to temperature sensitiv-ity with dnaN159 by selection for the nearby tnaA300::Tn10allele. The results of this experiment indicated that both strainscould be efficiently transduced with dnaN159, demonstrating

FIG. 2. Steady-state levels of the different umuD gene products inrecA, recA441, and recA730 strains. E. coli strains were grown in LBmedium to an OD

595 of 0.5. A 2-ml aliquot of each culture wascollected by centrifugation. Cells were resuspended in 20 l of sterile0.8% NaCl and lysed by addition of 50 l of 4X sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) loading dye (200mM Tris-HCl [pH 6.8], 8% SDS, 0.4% bromophenyl blue, 40% glyc-erol) containing 10% -mercaptoethanol prior to visualization byWestern blot analysis using purified polyclonal antibodies specific tothe UmuD and UmuD proteins. The salient features of each strainsgenotype are indicated. Strain examined: MS104, dnaN recA recR;MS105, dnaN159 recA recR; RM112, dnaN recA441 recR; RM113,

dnaN159 recA441 recR; RM133, dnaNrecA730 recR252::Tn109kan;RM134, dnaN159 recA730 recR252::Tn109kan; RM115, dnaN

recA730 recR; RM129, dnaN159 srd-1 recA730 recR; and RM130,dnaN159 srd-2 recA730 recR. Levels of UmuD and UmuD in strainsRM110 [dnaN lexA51(Def) uvrA6] and RM111 [ dnaN159 lexA51(Def)uvrA6] were essentially identical to those observed in strains MS104 andMS105, respectively (data not shown).

TABLE 3. Genetic requirements for dnaN159 recA730 synthetic lethality

StrainaRelevant genotypeb Transduction efficiencyc

recA lexA OtherNo. of transductants selected

(Tcr or Kmr CFU)No. ts/total no. screened

(% of total)

RW540 51(Def) 94 40/48 (83)RM112 441 51(Def) 89 41/50 (82)

RW576 730 51(Def) 552 2/94 (2)e

RM122 730 51(Def) polB 1,227 59/98 (60)RM116 730 51(Def) dinB 37 0/35 (3)RM118 730 51(Def) umuDC 65 5/11 (46)RM126d 730 51(Def) polB, umuDC 616 42/52 (81)RM135e 730 51(Def) srd-1 920 77/100 (77)RM136e 730 51(Def) srd-2 2,008 64/100 (64)RM132 730 51(Def) recR 58 8/20 (40)

a Strains are described in Table 1.bAbbreviations: , wild type; 441, recA441; 730, recA730; 51(Def), lexA51(Def); polB (Pol II), (araD-polB)::; dinB (Pol IV), (dinB-yafN)::kan; umuDC

(Pol V), umuDC595:: cat; srd-1 and srd-2, suppressors of recA730 dnaN159 synthetic lethality; and recR, recR252::Tn109kan.c Transductions were performed using a lysate propagated on a dnaN159 tnaA300::Tn10 strain except for strains RM112 and RM126, which used a lysate propagated on

adnaN159 zid-3162::Tn10kan strain(due to thefact that they containedthesrlC300::Tn10 andfadR613::Tn10 alleles, respectively). The efficiency of cotransduction ofdnaN159and tnaA300::Tn10 (or zid-3162::Tn10kan) was measured by patching out transductants in duplicate, followed by incubation overnight at 30 and 42C. Ratios represent thenumber of temperature-sensitive (ts) transductants divided by the number of clones patched out. Cotransduction frequencies for tnaA300::Tn10-dnaN159 (orzid-3162::Tn10kan-dnaN159) are indicated. Theoretical cotransduction frequencies were calculated to be 78% for tnaA300::Tn10 and dnaN, and 81% for zid-3162::Tn10kanand dnaNusing the following formula: F (1 d/L)3, where Fis the frequency of cotransduction, d is the distance between dnaNand zid or tnaA, and L is the size of the

fragment that can be transduced by P1 vir(2.1 min) (40).d Transduction of the umuDC595::cat allele into recA730 (araD-polB):: strain RM122 was inefficient (data not shown), suggesting that Pol II or Pol V was required by

(or at least important to) the recA730 strain. Therefore, we constructed a umuDC595::cat fadR613::Tn10 strain (RM125) so that we could measure the efficiency ofcotransduction of umuDC and fadR using the recA730 (araD-polB):: strain (RM122) and selection for Tcr. Cotransduction frequency for umuDC595::cat andfadR613::Tn10 was calculated to be 85%. Our finding that 39 of 45 (86%) Tc r transductants tested (from a total of 119 Tcr transductants) were also Cmr was in excellentagreement with the theoretical value of 85%, indicating that neither Pol II nor Pol V is required by the recA730 strain for viability.

e The two tetracycline-resistant and temperature-sensitive dnaN159 recA730 strains identified were colony purified and named RM129 and RM130. The dnaN159 allele instrains RM129 and RM130 was replaced by dnaN using a P1vir lysate grown on the dnaN zid-3162::Tn10kan strain. Three hundred eighty-four Kmr transductants wereobtained with RM129, while 408 were obtained with RM130. Thirty-nine of 40 tested for RM129 were temperature resistant, and 35 of the 39 temperature-resistant cloneswere Tcs. Thirty-eight of 40 tested for RM130 were temperature resistant, and 31 of the 38 temperature-resistant clones were Tc s. Four temperature-resistant and Tcs clonesfor each strain were picked and colony purified. One of each colony-purified strain was selected (named RM135 and RM136; see Table 1) for transduction with a P1 vir lysategrown on the dnaN159 tnaA300::Tn10 strain, the results of which are shown above.


8/2/2019 RecA-SOS

7/12

that each of these strains contained at least one mutation thatdid not map within polB or umuDC and was unlinked to dnaNthat acted to suppress the lethal effects of dnaN159 in therecA730 strain (Table 3).

The fact that strains RM129 and RM130 were viable butlacked mutations in polB and umuDC indicated that lethalitycould be suppressed by mutations affecting genes other thanPol II and Pol V. The RecF, RecO, and RecR proteins havebeen shown to play important roles in enhancing RecA func-tion (37). We therefore asked whether the recR252::Tn109kanallele would allow efficient construction of a recA730 dnaN159strain. As shown in Table 3, dnaN159 was transduced effi-ciently into the recA730 recR252::Tn109kan strain. None-theless, nucleotide sequence analysis of the recF, recO, andrecR genes from strains RM129 and RM130 revealed that eachcontained the correct sequence, indicating that suppression inthese two strains was conferred by a mutation affecting a dif-ferent gene(s).

In order to better understand the basis for lethality of therecA730 dnaN159 strain, as well as the suppression of lethalityby the recR252::Tn109kan, srd-1, and srd-2 alleles, we in- vestigated whether these alleles exerted an effect upon PolV-dependent UV-induced and/or spontaneous mutagenesis.

It is known that RecA is required for catalytic activity of PolV (48, 50, 62, 64). Thus, ifrecR252::Tn109kan, srd-1, and/orsrd-2 impairs RecA function, Pol V function may also be im-paired. Consistent with this hypothesis, the dnaN anddnaN159 recR252::Tn109kan strains displayed 5-fold and2-fold lower UV-induced mutation rates, respectively, com-pared to the dnaNrecR parent (Fig. 3A). Since these strainscontained the lexA51(Def) allele, these results demonstrate animportant role for the RecR protein in Pol V-dependent mu-tagenesis independent of SOS induction. Consistent with thisfinding, spontaneous mutation rates were modestly reduced inboth the dnaN and dnaN159 recR252::Tn109kan strains(Fig. 3B). Thus, the phenotype of the dnaN159 recR252::Tn109kan strain is consistent with a model in which dele-tion of recR suppresses synthetic lethality by impairing Pol Vfunction.

In order to determine whether srd-1 or srd-2 mediated sup-pression via an effect on Pol V function, we measured UV-induced SOS mutagenesis of strains RM129 and RM130. PolV-dependent UV-induced mutagenesis was reduced 5-foldin strain RM129 (srd-1) relative to the dnaN recA730 controlstrain RM115 (Fig. 3A). In contrast, proficiency in SOS mu-tagenesis of strain RM130 (srd-2) was similar to that observed

for RM115 (Fig. 3A). We also measured spontaneous mu-tagenesis frequencies in both the dnaN159 recA730 srd-1(RM129) and dnaN159 recA730 srd-2 (RM130) strains. Whilestrains RM130 (srd-2) and RM115 displayed similar spontane-ous mutation frequencies, spontaneous mutagenesis in strainRM129 (srd-1) was elevated more than sixfold relative to thednaN control strain RM115 (Fig. 3B). Furthermore, this el-evated mutation frequency was completely dependent uponPol V, as the umuDC595::cat allele eliminated it (Fig. 3B).These results indicate that suppression of the dnaN159recA730 synthetic lethality can be achieved simply by attenu-ating access to the replication fork of Pol V rather than affect-ing the catalytic activity of the enzyme per se. Based on these

FIG. 3. UV-induced and spontaneous mutation rates in differentdnaN and dnaN159 lexA51(Def) recA730 strains. UV-induced(A) and spontaneous (B) mutagenesis frequencies were measured asdescribed in Materials and Methods. Values presented are relative tothe dnaN recA730 strain (RM115), which was set equal to 100%.Strain RM115 yielded 44.8 Rifr CFU/107 survivors for UV-inducedmutagenesis and 2.2 Rifr CFU/107 survivors for spontaneous mutagen-esis. Other strains examined: RM123, dnaN recA730 (araD-polB)::;RM124, dnaN159 recA730 (araD-polB)::; RM119, dnaN recA730umuDC595::cat; RM120, dnaN159 recA730 umuDC595::cat; RM133,

dnaN recA730 recR252::Tn109kan; RM134, dnaN159 recA730recR252::Tn109kan; RM129, dnaN159 recA730 srd-1; RM130, dnaN159

recA730 srd-2; andRM131,dnaN159 recA730 srd-1 umuDC595::cat. Resultsshown are averages of at least three experiments standard deviation.


8/2/2019 RecA-SOS

8/12

results, we conclude that the srd-1 allele in strain RM129 affectsthe way in which Pol V is recruited to the replication fork.

Functionally distinct roles for Pol II and Pol V in conferring

recA730 dnaN159 synthetic lethality. In order to determinewhether the dnaN159 recA730 synthetic lethality required theconcerted actions of Pol II and Pol V or whether lethalityresulted from the combined effects of their independent ac-tions, we measured proficiency in Pol V-dependent SOSmutagenesis of the dnaN and dnaN159 (araD-polB)::strains. As shown in Fig. 3, the frequencies of UV-inducedand spontaneous mutagenesis in the dnaN and dnaN159(araD-polB):: strains were within twofold of each other.Thus, taken together, these results indicate that Pol II does notsignificantly affect Pol V function in translesion DNA synthe-sis, suggesting that synthetic lethality of the dnaN159 recA730strain results from the combined effects of Pol II and Pol V andnot from their concerted actions.

To rule out the possibility that Pol II and Pol V were work-ing together in some facet of replication that did not result in

an increased mutation frequency, but did confer lethality in therecA730 strain, we also examined the growth rates of the dif-ferent recA730 strains. The rapid growth rate of the dnaN159(araD-polB):: recA730 lexA51(Def) strain (RM124) sug-gests that inactivation of Pol II resulted in efficient suppressionof synthetic lethality (Fig. 4). In contrast, the very slow growthrate of the dnaN159 umuDC595:: cat recA730 lexA51(Def)strain (RM120) suggests that either (i) suppression of lethalityby inactivation of Pol V is inefficient or (ii) despite its ability toconfer lethality in the dnaN159 recA730 genetic background,Pol V nonetheless plays one or more important roles in thednaN159 strain. This latter possibility was suggested by ourprevious observation that the umuDC gene products play animportant role in protecting the dnaN159 mutant from thelethal effects of UV irradiation (56).

In order to distinguish between these two possibilities, weexamined the growth properties of the dnaN and dnaN159 recA730 lexA51(Def) strains bearing both the polB and theumuDC alleles. The results of this analysis indicated that

FIG. 4. Growth rates of different dnaN and dnaN159 lexA51(Def) recA730 strains. Overnight cultures of E. coli strains grown in LB mediumwere subcultured into the same medium to a starting OD

595of 0.04. Cultures were grown at 30C with shaking, and the OD

595was measured and

recorded every 20 min. Strains examined: RM115, dnaN recA730; RM123, dnaN recA730 (araD-polB)::; RM124, dnaN159 recA730(araD-polB)::; RM119, dnaN recA730 umuDC595::cat; RM120, dnaN159 recA730 umuDC595::cat; RM133, dnaN recA730 recR252::Tn109kan; RM134, dnaN159 recA730 recR252::Tn109kan; RM129, dnaN159 recA730 srd-1; RM130, dnaN159 recA730 srd-2; and RM131,

dnaN159 recA730 srd-1 umuDC595::cat. Results shown represent the average of triplicates standard deviation.


8/2/2019 RecA-SOS

9/12

inactivation of Pol II largely suppressed the poor growth ofthe dnaN159 umuDC595:: cat recA730 lexA51(Def) strain(Fig. 4, inset). Thus, these findings are consistent with amodel in which Pol V competes with Pol II for access to thereplication fork and that inactivation of Pol V alone sup-presses synthetic lethality less efficiently than does inactiva-tion of Pol II.

We also examined the growth rates of the recR, srd-1, andsrd-2 strains in order to compare their efficiency of suppressionto those observed for the polB and umuDC strains. Ourfinding that strain RM134 ( dnaN159 recA730 recR252::Tn109kan) grew almost as well as the dnaN recA730 strain(Fig. 4) suggests that, in addition to impairing Pol V function,the recR252::Tn109kan allele also affects Pol II function,presumably by impairing RecA730 activity. Both the srd-1(RM129) and srd-2 (RM130) strains grew as efficiently as thestrain carrying the (araD-polB):: allele, suggesting thatthese mutations were effective suppressors of synthetic lethal-ity (Fig. 4).

recA730 allele suppresses UV sensitivity of the dnaN159

strain. The model that begins to emerge from our resultsdiscussed above is that although transcriptional derepressionof the SOS regulon appears to play an important role in al-lowing Pol IV to gain access to the replication fork, RecAprotein subsequently facilitates access of Pol II and Pol V. If,in the course of promoting access of Pol II and Pol V to thefork, RecA mitigates access of Pol IV, either actively or as aresult of increased competition with Pol II and Pol V, then itfollows that the recA730 allele would suppress the UV sensi-tivity of the dnaN159 strain, much the same as the(dinB-yafN)::kan allele did (functional Pol IV, encoded bydinB, conferred UV sensitivity upon the dnaN159 strain [56]).

As a test of this hypothesis, we measured the UV sensitivity

of the dnaN159 recA730 srd-1 (RM129) and srd-2 (RM130)strains as well as the dnaN recA730 srd parent, RM115 (wewere unable to examine the UV sensitivity of the dnaN159recA730 srd strain due to the fact that it was inviable). Ashypothesized, RM129 and RM130 were each indistinguishablefrom the dnaN parent strain with respect to UV sensitivity(Fig. 5). Although we do not yet know the nature of the srd-1and srd-2 alleles, and hence we do not know the effect(s) thatthese mutations may have on UV sensitivity, these results arenonetheless consistent with a model in which RecA, eitherdirectly or indirectly, plays a role in attenuating access of PolIV to the replication fork as part of the global SOS response.Consistent with this conclusion, Pol IV is reportedly ineffi-cient at replicating a RecA-coated DNA template in vitro(32, 63, 67).

DISCUSSION

The steady-state levels and relative affinities of the variousE. coli DNA polymerases for the clamp, as well as possiblyother proteins that help to guide them to the replication fork,have presumably been optimized through evolution to ensurean ideal balance between accurate replication and capacity fortranslesion DNA synthesis. The goal of the work discussed inthis report was to better understand the roles of LexA andRecA in mediating this process. We hypothesized that sincePol II, Pol IV, and Pol V are all SOS regulated, the global SOS

response might act to help manage the actions of these poly-merases by regulating their access to the replication fork. As a

test of this hypothesis, we used different lexA and recA allelesto determine the individual effects of SOS induction and RecAactivation on Pol usage in the dnaN159 mutant.

Our results indicate that inactivation of LexA (Table 2) ortransformation with a plasmid directing expression of Pol IV(Fig. 1) is sufficient for this polymerase to impair growth of thednaN159 strain. In contrast, both SOS induction and activatedRecA protein, provided by the recA730 allele, were required inorder for Pol II or Pol V to impair growth of the dnaN159strain (Table 3). The fact that RecA730 is chronically activedue to a mutation and therefore cannot return to a restingstate appears to be vital for its synthetic lethality with dnaN159.Thus, our inability to observe a similar phenotype in a recA

dnaN159 strain is presumably due to the fact that RecA istransiently activated in response to DNA damage and activatedRecA levels dissipate as the damage is repaired or tolerated.Nonetheless, we have recently determined that the UV sensi-tivity of the dnaN159 umuDC strain is suppressed by inacti-vation of Pol II (M. D. Sutton and J. M. Duzen, submitted),indicating that Pol V and Pol II do confer a conditionally lethalphenotype in the recA dnaN159 strain following UV irradia-tion. Taken together, these results suggest that RecA protein,either directly or indirectly, influences the ability of the differ-ent SOS-regulated polymerases to gain access to the replica-tion fork in vivo (Fig. 6).

Our finding that recA730 suppressed the UV sensitivity ofthe dnaN159 strain (Fig. 5) is consistent with a model in which

FIG. 5. UV sensitivity ofE. coli srd-1 and srd-2 strains RM129 and

RM130. Overnight cultures of strains RM115 (dnaN

recA730),RM129 ( dnaN159 recA730 srd-1), and RM130 ( dnaN159 recA730

srd-2) grown in M9 minimal medium supplemented with glucose(0.2%), thiamine (1 g/ml), and Casamino Acids (0.5%) were subcul-tured into the same medium and grown at 30C with shaking untilreaching an OD

595of 0.6. A 5-ml aliquot of each culture was irra-

diated as indicated previously (57), and appropriate dilutions of eachirradiated and unirradiated sample were plated onto LB plates andincubated for two days at 30C. Results shown are the average of twoindependent experiments the range.


8/2/2019 RecA-SOS

10/12

RecA can impair access to the fork of Pol IV. However, itshould be stressed that Pol IV can still presumably competewith Pol II and Pol V for access to the replication fork in thepresence of activated RecA protein: indeed, the (dinB-yafN)::kan allele modestly enhanced Pol V-dependent UV-inducedmutagenesis of a recA dnaN159 strain (data not shown), con-sistent with a model in which the RecA protein can act toattenuate competition between Pol IV and polymerases II andV. Thus, our findings suggesting that the duration and the degreeof SOS induction influences polymerase selection (Fig. 6) couldexplain why the recA lexA holD::kan strain displayed aconditional lethality that was dependent upon Pol II, and to alesser extent, upon Pol IV (65), while a similar dnaN159 straindisplayed a conditional lethality dependent upon Pol IV (56):the holD::kan strain appears to be induced to a greater levelfor the SOS response than is the dnaN159 strain (56, 65),arguing that levels of activated RecA protein are higher. This,in turn, presumably results in the Pol II-dependent effect in theholD::kan strain. The inability of Pol V to impair growth of

the holD::kan strain may be due to the fact that activatedRecA levels were insufficient to allow the accumulation of asufficient steady-state level of Pol V to impair growth. Ourfinding that Pol II was more effective than Pol V at impairinggrowth of the dnaN159 recA730 strain (Fig. 4) is consistent withthis model.

Our finding that deletion of either polB (Pol II) or umuDC(Pol V) suppressed the lethality of the dnaN159 recA730 strainsuggests that Pol II and Pol V are able to gain access to thereplication fork in a RecA-mediated fashion. Our findings thatdeletion ofpolB suppressed the growth defect of the dnaN159recA730 umuDC595::cat strain (Fig. 4), and conferred a 2-fold effect on Pol V-dependent mutagenesis in the recA730strain (Fig. 3), irrespective of the dnaNallele, suggest that PolII and Pol V compete with each other for access to the repli-cation fork. Consistent with this conclusion, deletion of polB inthe dnaN159 lexA51(Def) recA strain (RM105) resulted in a17-fold increase in spontaneous argE3(Oc)3Arg reversion(data not shown). Subsequent deletion of umuDC confirmedthat spontaneous mutagenesis was Pol V dependent, suggest-

ing that in the dnaN159 strain, Pol II competed effectively withPol V for access to the replication fork, effectively suppressingmutagenesis. The fact that Pol V requires RecA for catalyticactivity indicates that RecA was activated in the cells thatdisplayed Pol V-dependent spontaneous mutagenesis, consis-tent with our genetic analyses suggesting that RecA effects Polselection.

Given that 159 is impaired for interaction with the cat-alytic subunit of Pol III (56) and that lagging-strand Pol IIImust cycle to a new primer every 1 second during lagging-strand synthesis (39), it is possible that the conditional lethalityof Pol IV in the dnaN159 lexA51(Def) strain is due to compe-tition of Pol IV with the lagging-strand polymerase, impairing

Okazaki fragment synthesis. Alternatively, Pol IV might com-pete with Pol I to impair Okazaki fragment maturation and/orssDNA gap repair. Our observation that Pol I is essential in thednaN159 strain (56) is consistent with the idea that this straindisplays an increased dependence upon ssDNA gap repair.Hence, competition between Pol IV and Pol I and/or Pol IIIfor access to nascent Okazaki fragments might result in per-sistent ssDNA gaps in the lagging strand. These gaps, in turn,could allow the formation of RecA/ssDNA nucleoprotein fil-aments, leading to the chronic, low-level SOS induction ob-served in the dnaN159 strain (56). Pol II and Pol V mightsimilarly compete with Pol III and Pol I for nascent Okazakifragments, resulting in ssDNA gaps. However, our observationthat the umuDC595::cat allele did not suppress the lethalityof the dnaN159 polA::kan strain (data not shown) indicatesthat the polA::kan and recA730 alleles affect the dnaN159strain in different ways.

It was recently reported that dinB transcription is induced inE. coli by -lactam-mediated inhibition of cell wall synthesis ina lexA- and recA-independent manner (35, 44). Importantly,-lactam-mediated transcriptional induction ofdinB correlateswith an increase in the frequency of 1 lacZ frameshift mu-tagenesis in vivo (44). This finding is consistent with our re-sults, suggesting that, in the dnaN159 strain, access to thereplication fork of Pol IV is regulated largely by its expressionlevel (although our results do not rule out the possibility thatadditional SOS-regulated gene products may be required for

FIG. 6. Model to describe the role of the E. coli sliding clamp,RecA protein and the global SOS response in managing polymeraseusage at the replication fork. (A) Summary of the phenotypes observedfor the dnaN159 mutant strain. Transcriptional de-repression of theSOS regulon by the lexA51(Def) allele leads to a conditional growthdefect of the dnaN159 strain that is suppressed by inactivation of the

dinB-encoded Pol IV. Subsequent introduction of the recA730 alleleinto the dnaN159 lexA51(Def) strain leads to a synthetic lethal pheno-type that is suppressed by inactivation of the polB-encoded Pol II orthe umuDC-encoded Pol V. (B) Model to describe the role of the SOSresponse in polymerase selection and switching in E. coli. Polymerasesin bold are proposed to have the ability to gain access to the replicationfork under the indicated conditions, and hence play prominent roles inDNA replication, while those in italics, although present, are proposed

to play more minor roles. The lack of a polymerase indicates that it isnot expressed to a significant level (or is not active) under the indicatedconditions. UmuD

2C participates in DNA damage checkpoint control

and is an inactive precursor to Pol V (38, 43). RecA/ssDNA-facilitatedautodigestion of UmuD to yield UmuD serves to release the check-point while simultaneously activating Pol V (UmuD2C) for translesionDNA synthesis. See the text for details.


8/2/2019 RecA-SOS

11/12

this process; see Table 1 and Fig. 1). The finding that Pol IV isexpressed at 6 to 12 times higher steady-state levels than theother SOS-regulated polymerases is consistent with the ideathat it is able to outcompete the other polymerases for bindingto the clamp and subsequent access to the replication fork(23). This conclusion is further supported by reports that mod-est overexpression of Pol IV from a multicopy plasmid signif-icantly increases the frequency of untargeted mutagenesis, in-dicating Pol IV-dependent replication (22).

Thus, E. coli appears to utilize different control systems tomanage the actions of each of its three SOS-regulated poly-merases: the actions of Pol IV appear to be largely regulated bytranscriptional controls, while the actions of Pol II and Pol Vappear to be regulated in a far more complex manner: in thecase of Pol V, these controls are incredibly complex and rangefrom RecA/ssDNA-mediated autodigestion of UmuD to yieldUmuD (7, 45, 53) to ClpXP- and Lon-mediated proteolysis ofPol V (14, 17, 18).

In conclusion, our results indicate that the global SOS re-sponse plays important roles in helping to manage the actions

of Pol II, Pol IV, and Pol V. Furthermore, our findings suggestthat RecA protein is able to attenuate the function of Pol IIand Pol V as well as possibly Pol III and Pol IV in vivo. Furthercharacterization of the roles of RecA protein in polymerasefunction as well as the identification of the srd-1 and srd-2 geneproducts will lead to a better understanding of polymerasemanagement in E. coli.

ACKNOWLEDGMENTS

We thank Mary Berlyn (E. coli Genetic Stock Center, Yale Univer-sity) for advice regarding the nomenclature for the suppressor muta-tions in strains RM129 and RM130 and Roger Woodgate (NICHD,NIH) for his generous gift of antibodies specific to the UmuD/UmuD proteins. We also thank Mary Berlyn, Justin Courcelle (Portland State

University), Benedicte Michel (Institut National de la Recherche Agronomique), Graham Walker (Massachusetts Institute of Technol-ogy), and Roger Woodgate for generously providing E. coli strains,Laurie Sanders for her comments on the manuscript, and the membersof our laboratory for helpful discussions.

This work was supported by Public Service Health grant GM066094to M.D.S.

REFERENCES

1. Ason, B., J. G. Bertram, M. M. Hingorani, J. M. Beechem, M. ODonnell,M. F. Goodman, and L. B. Bloom. 2000. A model for Escherichia coli DNApolymerase III holoenzyme assembly at primer/template ends. DNA triggersa change in binding specificity of the gamma complex clamp loader. J. Biol.Chem. 275:30063015.

2. Becherel, O. J., R. P. Fuchs, and J. Wagner. 2002. Pivotal role of the -clampin translesion DNA synthesis and mutagenesis in E. coli cells. DNA Repair(Amsterdam) 1:703708.

3. Bertram, J. G., L. B. Bloom, M. ODonnell, and M. F. Goodman. 2004.

Increased dNTP binding affinity reveals a nonprocessive role for Escherichiacoli beta clamp with DNA polymerase IV. J. Biol. Chem. 279:3304733050.

4. Bonner, C. A., S. Hays, K. McEntee, and M. F. Goodman. 1990. DNApolymerase II is encoded by the DNA damage-inducible dinA gene ofEsch- erichia coli. Proc. Natl. Acad. Sci. USA 87:76637667.

5. Bonner, C. A., P. T. Stukenberg, M. Rajagopalan, R. Eritja, M. ODonnell,K. McEntee, H. Echols, and M. F. Goodman. 1992. Processive DNA synthe-sis by DNA polymerase II mediated by DNA polymerase III accessoryproteins. J. Biol. Chem. 267:1143111438.

6. Bowman, G. D., E. R. Goedken, S. L. Kazmirski, M. ODonnell, and J.Kuriyan. 2005. DNA polymerase clamp loaders and DNA recognition. FEBSLett. 579:863867.

7. Burckhardt, S. E., R. Woodgate, R. H. Scheuermann, and H. Echols. 1988.UmuD mutagenesis protein ofEscherichia coli: overproduction, purification,and cleavage by RecA. Proc. Natl. Acad. Sci. USA 85:18111815.

8. Burgers, P. M., A. Kornberg, and Y. Sakakibara. 1981. The dnaNgene codesfor the subunit of DNA polymerase III holoenzyme of Escherichia coli.Proc. Natl. Acad. Sci. USA 78:53915395.

9. Courcelle, J., C. Carswell-Crumpton, and P. C. Hanawalt. 1997. recF andrecR are required for the resumption of replication at DNA replication forksin Escherichia coli. Proc. Natl. Acad. Sci. USA 94:37143719.

10. Courcelle, J., A. Khodursky, B. Peter, P. O. Brown, and P. C. Hanawalt.2001. Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 158:4164.

11. Cox, M. M., M. F. Goodman, K. N. Kreuzer, D. J. Sherratt, S. J. Sandler,and K. J. Marians. 2000. The importance of repairing stalled replicationforks. Nature 404:3741.

12. Dalrymple, B. P., K. Kongsuwan, G. Wijffels, N. E. Dixon, and P. A.Jennings. 2001. A universal protein-protein interaction motif in the eubac-terial DNA replication and repair systems. Proc. Natl. Acad. Sci. USA98:1162711632.

13. Fijalkowska, I. J., R. L. Dunn, and R. M. Schaaper. 1997. Genetic require-ments and mutational specificity of the Escherichia coli SOS mutator activity.J. Bacteriol. 179:74357445.

14. Frank, E. G., D. G. Ennis, M. Gonzalez, A. S. Levine, and R. Woodgate. 1996.Regulation of SOS mutagenesis by proteolysis. Proc. Natl. Acad. Sci. USA93:1029110296.

15. Friedberg, E. C., R. Wagner, and M. Radman. 2002. Specialized DNApolymerases, cellular survival, and the genesis of mutations. Science 296:16271630.

16. Friedberg, E. C., G. C. Walker, and W. Siede. 1995. DNA repair and mu-tagenesis. ASM Press, Washington, D.C.

17. Gonzalez, M., E. G. Frank, A. S. Levine, and R. Woodgate. 1998. Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: invitro degradation and identification of residues required for proteolysis.Genes Dev. 12:38893899.

18. Gonzalez, M., F. Rasulova, M. R. Maurizi, and R. Woodgate. 2000. Subunit-specific degradation of the UmuD/D heterodimer by the ClpXP protease:the role of trans recognition in UmuD stability. EMBO J. 19:52515258.

19. Grompone, G., M. Seigneur, S. D. Ehrlich, and B. Michel. 2002. Replicationfork reversal in DNA polymerase III mutants of Escherichia coli: a role forthe clamp. Mol. Microbiol. 44:13311339.

20. Hingorani, M. M., and M. ODonnell. 1998. ATP binding to the Escherichiacoli clamp loader powers opening of the ring-shaped clamp of DNA poly-merase III holoenzyme. J. Biol. Chem. 273:2455024563.

21. Ho, C., O. I. Kulaeva, A. S. Levine, and R. Woodgate. 1993. A rapid methodfor cloning mutagenic DNA repair genes: isolation of umu-complementinggenes from multidrug resistance plasmids R391, R446b, and R471a. J. Bac-teriol. 175:54115419.

22. Kim, S. R., G. Maenhaut-Michel, M. Yamada, Y. Yamamoto, K. Matsui, T.Sofuni, T. Nohmi, and H. Ohmori. 1997. Multiple pathways for SOS-inducedmutagenesis in Escherichia coli: an overexpression of dinB/dinP results in

strongly enhancing mutagenesis in the absence of any exogenous treatmentto damage DNA. Proc. Natl. Acad. Sci. USA 94:1379213797.23. Kim, S. R., K. Matsui, M. Yamada, P. Gruz, and T. Nohmi. 2001. Roles of

chromosomal and episomal dinB genes encoding DNA pol IV in targetedand untargeted mutagenesis in Escherichia coli. Mol. Genet. Genomics 266:207215.

24. Kowalczykowski, S. C., D. A. Dixon, A. K. Eggleston, S. D. Lauder, andW. M. Rehrauer. 1994. Biochemistry of homologous recombination in Esch- erichia coli. Microbiol. Rev. 58:401465.

25. Lavery, P. E., and S. C. Kowalczykowski. 1992. Biochemical basis of theconstitutive repressor cleavage activity of recA730 protein. A comparison torecA441 and recA803 proteins. J. Biol. Chem. 267:2064820658.

26. Lavery, P. E., and S. C. Kowalczykowski. 1990. Properties of recA441 pro-tein-catalyzed DNA strand exchange can be attributed to an enhanced abilityto compete with SSB protein. J. Biol. Chem. 265:40044010.

27. Lenne-Samuel, N., J. Wagner, H. Etienne, and R. P. Fuchs. 2002. Theprocessivity factor controls DNA polymerase IV traffic during spontaneousmutagenesis and translesion synthesis in vivo. EMBO Rep. 3:4549.

28. Lopez de Saro, F. J., R. E. Georgescu, M. F. Goodman, and M. ODonnell.2003. Competitive processivity-clamp usage by DNA polymerases duringDNA replication and repair. EMBO J. 22:64086418.

29. Lopez de Saro, F. J., and M. ODonnell. 2001. Interaction of the beta slidingclamp with MutS, ligase, and DNA polymerase I. Proc. Natl. Acad. Sci. USA98:83768380.

30. Lusetti, S. L., and M. M. Cox. 2002. The bacterial RecA protein and therecombinational DNA repair of stalled replication forks. Annu. Rev. Bio-chem. 71:71100.

31. Maki, S., and A. Kornberg. 1988. DNA polymerase III holoenzyme ofEsch- erichia coli. III. Distinctive processive polymerases reconstituted from puri-fied subunits. J. Biol. Chem. 263:65616569.

32. Maor-Shoshani, A., K. Hayashi, H. Ohmori, and Z. Livneh. 2003. Analysis oftranslesion replication across an abasic site by DNA polymerase IV of Esch- erichia coli. DNA Repair (Amsterdam) 2:12271238.

33. Maor-Shoshani, A., and Z. Livneh. 2002. Analysis of the stimulation of DNApolymerase V of Escherichia coli by processivity proteins. Biochemistry 41:1443814446.


8/2/2019 RecA-SOS

12/12

34. McHenry, C. S. 2003. Chromosomal replicases as asymmetric dimers: studiesof subunit arrangement and functional consequences. Mol. Microbiol. 49:11571165.

35. Miller, C., L. E. Thomsen, C. Gaggero, R. Mosseri, H. Ingmer, and S. N.Cohen. 2004. SOS response induction by beta-lactams and bacterial defenseagainst antibiotic lethality. Science 305:16291631.

36. Miller, J. H. 1992. A short course in bacterial genetics: a laboratory manualand handbook for Escherichia coli and related bacteria. Cold Spring HarborLaboratory Press, Plainview, N.Y.

37. Morimatsu, K., and S. C. Kowalczykowski. 2003. RecFOR proteins loadRecA protein onto gapped DNA to accelerate DNA strand exchange: auniversal step of recombinational repair. Mol. Cell 11:13371347.

38. Murli, S., T. Opperman, B. T. Smith, and G. C. Walker. 2000. A role for theumuDC gene products of Escherichia coli in increasing resistance to DNAdamage in stationary phase by inhibiting the transition to exponentialgrowth. J. Bacteriol. 182:11271135.

39. Naktinis, V., J. Turner, and M. ODonnell. 1996. A molecular switch in areplication machine defined by an internal competition for protein rings. Cell84:137145.

40. Neidhardt, F. C., and R. Curtiss. 1996. Escherichia coli and Salmonella:cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C.

41. Nohmi, T., J. R. Battista, L. A. Dodson, and G. C. Walker. 1988. RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relation-ship between transcriptional derepression and posttranslational activation.Proc. Natl. Acad. Sci. USA 85:18161820.

42. Ohmori, H., M. Kimura, T. Nagata, and Y. Sakakibara. 1984. Structuralanalysis of the dnaA and dnaN genes of Escherichia coli. Gene 28:159170.

43.Opperman, T., S. Murli, B. T. Smith, and G. C. Walker.

1999. A model fora umuDC-dependent prokaryotic DNA damage checkpoint. Proc. Natl.Acad. Sci. USA 96:92189223.

44. Perez-Capilla, T., M. R. Baquero, J. M. Gomez-Gomez, A. Ionel, S. Martin,and J. Blazquez. 2005. SOS-independent induction of dinB transcription bybeta-lactam-mediated inhibition of cell wall synthesis in Escherichia coli.J. Bacteriol. 187:15151518.

45. Perry, K. L., S. J. Elledge, B. B. Mitchell, L. Marsh, and G. C. Walker. 1985.umuDC and mucAB operons whose products are required for UV light- andchemical-induced mutagenesis: UmuD, MucA, and LexA proteins sharehomology. Proc. Natl. Acad. Sci. USA 82:43314335.

46. Pham, P., J. G. Bertram, M. ODonnell, R. Woodgate, and M. F. Goodman.2001. A model for SOS-lesion-targeted mutations in Escherichia coli. Nature409:366370.

47. Pham, P., E. M. Seitz, S. Saveliev, X. Shen, R. Woodgate, M. M. Cox, andM. F. Goodman. 2002. Two distinct modes of RecA action are required forDNA polymerase V-catalyzed translesion synthesis. Proc. Natl. Acad. Sci.USA 99:1106111066.

48. Reuven, N. B., G. Arad, A. Maor-Shoshani, and Z. Livneh. 1999. The mu-tagenesis protein UmuC is a DNA polymerase activated by UmuD , RecA,and SSB and is specialized for translesion replication. J. Biol. Chem. 274:3176331766.

49. Reuven, N. B., G. Arad, A. Z. Stasiak, A. Stasiak, and Z. Livneh. 2001.Lesion bypass by the Escherichia coli DNA polymerase V requires assemblyof a RecA nucleoprotein filament. J. Biol. Chem. 276:55115517.

50. Reuven, N. B., G. Tomer, and Z. Livneh. 1998. The mutagenesis proteinsUmuD and UmuC prevent lethal frameshifts while increasing base substi-tution mutations. Mol. Cell 2:191199.

51. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

52. Schlacher, K., K. Leslie, C. Wyman, R. Woodgate, M. M. Cox, and M. F.Goodman. 2005. DNA polymerase V and RecA protein, a minimal muta-some. Mol. Cell 17:561572.

53. Shinagawa, H., H. Iwasaki, T. Kato, and A. Nakata. 1988. RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis. Proc. Natl.Acad. Sci. USA 85:18061810.

54. Strauss, B. S., R. Roberts, L. Francis, and P. Pouryazdanparast. 2000. Roleof the dinB gene product in spontaneous mutation in Escherichia coli with animpaired replicative polymerase. J. Bacteriol. 182:67426750.

55. Stukenberg, P. T., P. S. Studwell-Vaughan, and M. ODonnell. 1991. Mech-anism of the sliding beta-clamp of DNA polymerase III holoenzyme. J. Biol.Chem. 266:1132811334.

56. Sutton, M. D. 2004. The Escherichia coli dnaN159 mutant displays alteredDNA polymerase usage and chronic SOS induction. J. Bacteriol. 186:67386748.

57. Sutton, M. D., J. M. Duzen, and R. W. Maul. 2005. Mutant forms of theEscherichia coli sliding clamp that distinguish between its roles in replica-tion and DNA polymerase V-dependent translesion DNA synthesis. Mol.Microbiol. 55:17511766.

58. Sutton, M. D., M. F. Farrow, B. M. Burton, and G. C. Walker. 2001. Geneticinteractions between the Escherichia coli umuDC gene products and the processivity clamp of the replicative DNA polymerase. J. Bacteriol. 183:28972909.

59. Sutton, M. D., T. Opperman, and G. C. Walker. 1999. The Escherichia coliSOS mutagenesis proteins UmuD and UmuD interact physically with thereplicative DNA polymerase. Proc. Natl. Acad. Sci. USA 96:1237312378.

60. Sutton, M. D., B. T. Smith, V. G. Godoy, and G. C. Walker. 2000. The SOSresponse: recent insights into umuDC-dependent mutagenesis and DNAdamage tolerance. Annu. Rev. Genet. 34:479497.

61. Sutton, M. D., and G. C. Walker. 2001. Managing DNA polymerases: coor-dinating DNA replication, DNA repair, and DNA recombination. Proc.Natl. Acad. Sci. USA 98:83428349.

62. Tang, M., I. Bruck, R. Eritja, J. Turner, E. G. Frank, R. Woodgate, M.ODonnell, and M. F. Goodman. 1998. Biochemical basis of SOS-inducedmutagenesis in Escherichia coli: reconstitution of in vitro lesion bypass de-pendent on the UmuD2C mutagenic complex and RecA protein. Proc. Natl.Acad. Sci. USA 95:97559760.

63. Tang, M., P. Pham, X. Shen, J. S. Taylor, M. ODonnell, R. Woodgate, andM. F. Goodman. 2000. Roles of E. coli DNA polymerases IV and V inlesion-targeted and untargeted SOS mutagenesis. Nature 404:10141018.

64. Tang, M., X. Shen, E. G. Frank, M. ODonnell, R. Woodgate, and M. F.Goodman. 1999. UmuD2C is an error-prone DNA polymerase, Escherichiacoli pol V. Proc. Natl. Acad. Sci. USA 96:89198924.

65. Viguera, E., M. Petranovic, D. Zahradka, K. Germain, D. S. Ehrlich, and B.Michel. 2003. Lethality of bypass polymerases in Escherichia coli cells with adefective clamp loader complex of DNA polymerase III. Mol. Microbiol.50:193204.

66. Wagner, J., H. Etienne, R. Janel-Bintz, and R. P. Fuchs. 2002. Genetics ofmutagenesis in E. coli: various combinations of translesion polymerases (PolII, IV and V) deal with lesion/sequence context diversity. DNA Repair

(Amsterdam) 1:159167.67. Wagner, J., S. Fujii, P. Gruz, T. Nohmi, and R. P. Fuchs. 2000. The clamptargets DNA polymerase IV to DNA and strongly increases its processivity.EMBO Rep. 1:484488.

68. Wang, R. F., and S. R. Kushner. 1991. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichiacoli. Gene 100:195199.

69. Wijffels, G., B. P. Dalrymple, P. Prosselkov, K. Kongsuwan, V. C. Epa, P. E.Lilley, S. Jergic, J. Buchardt, S. E. Brown, P. F. Alewood, P. A. Jennings, and

N. E. Dixon. 2004. Inhibition of protein interactions with the 2 sliding clampof Escherichia coli DNA polymerase III by peptides from 2-binding pro-teins. Biochemistry 43:56615671.

70. Witkin, E. M., J. O. McCall, M. R. Volkert, and I. E. Wermundsen. 1982.Constitutive expression of SOS functions and modulation of mutagenesisresulting from resolution of genetic instability at or near the recA locus of Escherichia coli. Mol. Gen. Genet. 185:4350.

71. Woodgate, R. 1992. Construction of a umuDC operon substitution mutationin Escherichia coli. Mutat. Res. 281:221225.


reca-sos

Documents