JOURNAL OF BACTERIOLOGY, Aug. 2009, p. 4815–4823 Vol. 191, No. 150021-9193/09/$08.00�0 doi:10.1128/JB.01742-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Steric Gate Variants of UmuC Confer UV Hypersensitivity onEscherichia coli�

Brenna W. Shurtleff,1 Jaylene N. Ollivierre,1 Mohammad Tehrani,1Graham C. Walker,2 and Penny J. Beuning1,3*

Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 021151; Department of Biology,Massachusetts Institute of Technology, Cambridge, Massachusetts 021392; and Center for Interdisciplinary Research on

Complex Systems, Northeastern University, Boston, Massachusetts 021153

Received 12 December 2008/Accepted 18 May 2009

Y family DNA polymerases are specialized for replication of damaged DNA and represent a major contri-bution to cellular resistance to DNA lesions. Although the Y family polymerase active sites have fewer contactswith their DNA substrates than replicative DNA polymerases, Y family polymerases appear to exhibit speci-ficity for certain lesions. Thus, mutation of the steric gate residue of Escherichia coli DinB resulted in thespecific loss of lesion bypass activity. We constructed variants of E. coli UmuC with mutations of the steric gateresidue Y11 and of residue F10 and determined that strains harboring these variants are hypersensitive to UVlight. Moreover, these UmuC variants are dominant negative with respect to sensitivity to UV light. The UVhypersensitivity and the dominant negative phenotype are partially suppressed by additional mutations in theknown motifs in UmuC responsible for binding to the � processivity clamp, suggesting that the UmuC stericgate variant exerts its effects via access to the replication fork. Strains expressing the UmuC Y11A variant alsoexhibit decreased UV mutagenesis. Strikingly, disruption of the dnaQ gene encoding the replicative DNApolymerase proofreading subunit suppressed the dominant negative phenotype of a UmuC steric gate variant.This could be due to a recruitment function of the proofreading subunit or involvement of the proofreadingsubunit in a futile cycle of base insertion/excision with the UmuC steric gate variant.

All organisms are subject to exogenous and endogenousagents that damage DNA. DNA lesions can lead to polymerasestalling during replication and to mutagenesis, either of whichmay result in death of the cell (20). Y family DNA polymerasespossess a specialized ability to copy damaged DNA in a processknown as translesion synthesis and therefore represent a majorcontribution to DNA damage tolerance. However, Y familypolymerases copy undamaged DNA with an elevated error rateand thus may also contribute to mutagenesis (20).

Escherichia coli polymerase V (Pol V), the product of theumuDC genes, and Pol IV, the product of the dinB gene,belong to the Y family of DNA polymerases. These poly-merases are characterized by their ability to bypass lesions inDNA and by the low fidelity with which they replicate undam-aged DNA, potentially introducing mutations that may beharmful (20). E. coli dinB has thus been implicated in adaptivemutagenesis (8, 39, 56, 67), and both dinB and umuC appear tobe important in the development of resistance to antibiotics(11–14, 69). Therefore, the expression and activity of poten-tially mutagenic Y family DNA polymerases are necessarilytightly regulated. Y family polymerases in E. coli are regulatedas part of the SOS response (20), which is initiated by anaccumulation of single-stranded DNA (ssDNA) resulting froma cell’s efforts to replicate damaged DNA. RecA binds to thessDNA, forms a RecA:ssDNA nucleoprotein filament, andfacilitates the self-cleavage of LexA, the protein responsible

for transcriptional repression of the SOS regulon. The de-creased intracellular concentration of LexA results in induc-tion of at least 57 genes in E. coli, including genes encoding theY family DNA polymerases Pol IV and Pol V, which are ableto bypass a variety of lesions (20, 53, 54). E. coli Pol IV (DinB)efficiently and accurately copies DNA templates containingN2-furfuryl-dG lesions (29). E. coli Pol V (UmuD�2C) is able tocopy DNA templates containing abasic sites and the productsof UV irradiation, including thymine-thymine cyclobutane py-rimidine dimers and thymine-thymine (6-4) photoproducts,and is mutagenic when copying the latter (48, 65).

Once expressed, UmuC activity is regulated by the polymer-ase manager protein, UmuD. UmuD initially exists as theUmuD2 dimer; upon interaction with a RecA:ssDNA nucleo-protein filament, UmuD cleaves off its own N-terminal 24amino acids to form UmuD� (43). The UmuD�2 form is re-quired for UmuC to be active in translesion synthesis (48, 66).

In addition to the transcriptional and posttranslational reg-ulation of UmuC, another layer of regulation exists in that thepolymerase must gain access to the damaged DNA for lesionbypass to occur. While much remains to be understood aboutthe recruitment of polymerases in E. coli, protein-protein in-teractions, particularly with subunits of the replisome and withRecA, are important for regulating access of UmuC to thereplication fork (3, 4, 22, 26, 36, 50–52, 63). Whereas only a fewdirect physical interactions have been shown between UmuCand these proteins, there are numerous demonstrated interac-tions between the manager protein UmuD and/or UmuD� andother cellular proteins (9, 24, 40, 61, 64).

Interaction with the � clamp is thought to be integral to thecoordination of UmuC function (3, 4, 18, 62–64). The � pro-

* Corresponding author. Mailing address: Department of Chemistryand Chemical Biology, Northeastern University, 360 Huntington Av-enue, 102 Hurtig Hall, Boston, MA 02115. Phone: (617) 373-2865. Fax:(617) 373-8795. E-mail: beuning@neu.edu.

� Published ahead of print on 29 May 2008.

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cessivity clamp is a ring-shaped dimer that has one “hydropho-bic patch” per monomer to which the polymerases in E. colibind. All five E. coli DNA polymerases have been shown tobind to the � clamp, four of which bind via a QL[S/D]LF motif(15, 35). The � clamp has been shown to increase the proces-sivity of Pol III and Pol IV substantially, but the increase inprocessivity conferred on Pol V by the � clamp is relativelymodest (22, 36). Recent studies of bacteriophage T4 DNAreplication suggest a dynamic interaction in phage T4 in whichpolymerases are rapidly exchanged at the replication fork, aprocess facilitated by interactions with the processivity clamp(70). Moreover, it has been suggested that polymerase stallingmay be due to a futile cycle of base insertion and exonucleo-lytic proofreading at sites of DNA damage (46). Dynamic in-teractions at the replication fork could provide an opportunityfor alternate polymerases to be employed in lesion bypass andallow DNA replication to continue after damage (21).

DNA polymerases possess a steric gate residue near theactive site responsible for the ability of the polymerase toprevent the incorporation of ribonucleotides in DNA by block-ing the 2�-OH of incoming ribonucleotides (1, 23). The residueis conserved in Y family polymerases as either Tyr or Phe (Fig.1) (7, 17). In the case of E. coli DinB, mutation of the stericgate residue (F13V) resulted in an increase of rNTP incorpo-ration from a frequency of �10�5 to 10�3 (29). In the DinBsubfamily of Y family polymerases, including E. coli DinB andmammalian Pol �, the steric gate has been shown to be critical

for the specific bypass of DNA lesions (29). Mutation of thesteric gate residue (F13V) in E. coli DinB eliminated bypass ofN2-furfuryl-dG lesions but did not affect DNA polymeraseactivity on undamaged DNA (29). We show here that a singlesubstitution in the steric gate residue (Y11) of E. coli UmuCrenders cells hypersensitive to UV light and is dominant neg-ative with respect to sensitivity to UV and other DNA dam-aging agents. The dominant negative phenotype is suppressedby additional mutations within UmuC that disrupt binding tothe � clamp or by disruption of dnaQ, the proofreading subunitof the replicative DNA polymerase. We also show that UmuC-Y11A confers decreased UV mutability on cells that harborthis variant.

MATERIALS AND METHODS

Strains, plasmids, and general techniques. The strains and plasmids used inthis study are listed in Table 1. The o1c mutation present in the operator se-quence of both pGY9738 and pGY9739 is a single G3A mutation in the SOSconsensus box and results in twofold-higher expression levels of UmuD com-pared to plasmids harboring the o� operator sequence (60). However, expressionlevels of UmuD from o1

c plasmids in LexA� strains are lower than in strains thatlack LexA; thus, the o1

c mutation leads to only partial derepression (60). Stericgate (Y11A, Y11V, and F10V) and � clamp interaction mutants (313LTP315 toAAA and 357QLNLF361 to ALNAA) (4) of UmuC were constructed using theQuikChange site-directed mutagenesis kit (Stratagene). The mutations wereconfirmed by DNA sequence analysis (Massachusetts General Hospital CoreFacility, Cambridge, MA, and MIT Biopolymers Laboratory, Cambridge, MA).Competent cells were prepared using the CaCl2 method (49). Transformationsand immunoblotting were performed as described previously (5). Expressionlevels of UmuC were quantified using ImageQuant (GE Healthcare) by normal-izing to the cross-reacting band.

UV survival and mutagenesis assays. Selected colonies from fresh transfor-mations were grown overnight in LB and supplemented with spectinomycin (60�g/ml) or ampicillin (100 �g/ml). Dilutions (1:50) of the overnight cultures weregrown to an optical density at 600 nm (OD600) of 0.2 to 0.4. Equal numbers ofcells were suspended in 0.85% saline solution to an OD600 of 0.5. The cells werethen irradiated with UV light (254 nm) from a germicidal lamp (USHIO). Serialdilutions (1:10) were plated on 1.2% LB agar plates containing spectinomycin orampicillin and incubated at 37°C for 20 to 24 h. The data represent the averagesof at least three determinations, unless otherwise noted. Error bars represent thestandard deviations. Assays for sensitivity to nitrofurazone (5-nitro-2-furalde-hyde semicarbazone [NFZ]; Sigma-Aldrich) and methyl methanesulfonate(MMS; Sigma-Aldrich) were carried out by plating serial dilutions of transfor-mation mixtures on selective LB agar plates containing the indicated amount ofreagent and incubated at 37°C for 20 to 24 h.

Mutagenesis assays were performed as described previously (5). Briefly, cellswere irradiated at 10 J/m2 as described above and then plated on M9 minimalplates with trace amounts of arginine (1 �g/ml). Assays were carried out underconditions in which survival varied less than 11-fold (for the experiments sum-marized below in Fig. 6) or less than 4-fold (for experiments summarized belowin Fig. 8) across all samples and conditions, which was determined by plating onM9 minimal plates with 40 �g/ml arginine. CFU were determined after 48 h ofincubation at 37°C.

Growth assay. Selected colonies from fresh transformations were grown over-night in LB supplemented with spectinomycin (60 �g/ml). Dilutions of theovernight cultures were made to an OD600 of 0.05 and were grown at 37°C withshaking. From 0 h to 3 h samples were taken every half hour and the concen-tration assessed by measuring the OD600 and plating on 1.2% LB agar platessupplemented with spectinomycin. From 4 h to 8 h samples were taken everyhour and the concentration was determined by measuring the OD600.

RESULTS

Steric gate variants of UmuC render E. coli hypersensitive toUV light. The steric gate residue of DNA polymerases plays acritical role in preventing incorporation of ribonucleotides intoDNA (1, 23). In the case of E. coli DinB, the F13V steric gatevariant retained the ability to discriminate against ribonucle-

FIG. 1. The steric gate residue of UmuC is predicted to approachthe incoming nucleotide. (Top) Homology model of UmuC (4) show-ing the template (green), primer and incoming nucleotide (blue), andresidues F10 and steric gate Y11 (red). The backbone of UmuC isshown in yellow. The illustration was prepared using VMD (28). (Bot-tom) Amino acid sequences of representative Y family polymerasesshowing conserved residues aligned with F10 and Y11 of UmuC(boxed) (7). Ec, Escherichia coli; Sa, Sulfolobus acidocaldarius; Ss,Sulfolobus solfataricus; Sc, Saccharomyces cerevisiae; Hs, Homo sapiens.

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otides, albeit less proficiently than wild-type DinB (29). How-ever, it appears that, at least for E. coli DinB and its mamma-lian and archaeal ortholog, an additional role of the steric gateresidue is to facilitate lesion bypass (29). In order to assess therole in DNA damage tolerance of the steric gate in E. coliUmuC, we made alanine and valine mutations in UmuC atY11, as this residue is predicted to be the steric gate residuebased on sequence alignments (Fig. 1) (7). We also constructedUmuC F10V, as the position immediately N-terminal to thesteric gate is a conserved Phe residue that in the cases of E. coliUmuC (57) and the yeast Y family DNA polymerase Pol � (42)is important for lesion bypass. In most of these experiments,we employed a synthetic plasmid construct that expressesumuD�C, rather than umuDC, in order to eliminate any pos-sible confounding effects due to changes in UmuD cleavageefficiency (58). GW8017 (AB1157 umuDC) strains harboringlow-copy-number plasmids bearing UmuC variants were as-sayed for sensitivity to UV light. To our surprise, strains har-boring plasmids expressing the Y11A variant of UmuC notonly failed to complement the umuDC strain for UV-inducedmutagenesis but dramatically sensitized the cells to killing byUV light (Fig. 2). Strikingly, the UmuC Y11A variant rendersumuDC cells so sensitive to UV light that extreme killing isobserved at a dose of only 5 J/m2 (Fig. 2). A similar UV-sensitive phenotype was observed with the Y11A variant as-sayed in the context of pGY9739 (umuDC) (data not shown).The Y11V variant also caused UV hypersensitivity, whereasthe F10V variant caused more modest, but still striking, sen-sitivity (Fig. 2) comparable to that observed with a catalyticallyinactive UmuC variant (C104) (Fig. 2). As has been notedpreviously, loss of umuC has only a modest effect on UVsensitivity (20, 30), but here we found that a single point mu-tation in UmuC causes an increase in UV sensitivity of severalorders of magnitude. To the best of our knowledge, this is thefirst example of a point mutation in UmuC that renders cellssensitive to UV light to this extent.

The UmuC steric gate variant could confer UV hypersensi-

tivity either via a growth defect of the strain or by a specificdefect in polymerase or translesion synthesis activity. We as-sayed for growth the umuDC strain harboring a plasmid en-coding the UmuC Y11A steric gate variant. The growth ratesof the strains carrying plasmids encoding the UmuC steric gatevariant were indistinguishable from the wild type in the ab-sence of UV irradiation (Fig. 3A). We also confirmed that thesteady-state protein levels did not differ substantially betweenthe different constructs, as levels of UmuC varied approxi-mately twofold among the strains that harbor plasmids (Fig.3B). Approximately threefold more UmuC is expressed frompGY9738 (in GW8017) than from the chromosome inGW2771. Under these conditions, we did not detect UmuC inany of these samples in the absence of UV induction.

� clamp interaction motifs modulate the UV hypersensitiv-ity of the steric gate variant. We hypothesized that the UmuCY11A variant causes UV hypersensitivity because it can still be

TABLE 1. Strains and plasmids

Strain or plasmid Relevant genotype or description Reference or source

Bacterial strainsAB1157 argE3 umuDC� Laboratory stockGW8017 AB1157 umuDC 25GW2771 umuDC� 44GW2771 spq-2 GW2771 spq-2 44GW2771 spq-2 dnaQ903 dnaQ903::tet P1(RM3980)3GW2771 spq-2 (55)MS120 AB1157 dnaN59Ts 63PB101 AB1157 recJ P1(JW2860) 3 AB1157 (2)RW574 lexA51(Def) recA730 umuDC� 68RW598 lexA51(Def) recA730 umuDC� dnaE1026–3 proAB� 68DV14 lexA51(Def) recA730 umuDC� dnaE486 zae502::Tn10 68DV16 lexA51(Def) recA730 umuDC� dnaE9 zae502::Tn10 68DV17 lexA51(Def) recA730 umuDC� dnaE511 zae502::Tn10 68XL1 Blue Laboratory stock

PlasmidspGB2 Vector; pSC101 derived, Specr 10pGY9739 o1

C umuDC; pSC101 derived 58pGY9738 o1

C umuD�C; pSC101 derived 58pWSK30 Vector; pSC101 derived, Ampr 31pYG768 dinB� 31

FIG. 2. The steric gate Y11 and F10 variants in UmuC cause UVhypersensitivity. Assays were performed with the pGY9738 plasmidand the following derivatives in GW8017: pGY9738 (umuD�C; });pGB2 (empty vector; ); pGY9738-C104 (cat-) (umuD�C C104; �);pGY9738-F10V (umuD�C F10V; f); pGY9738-Y11V (umuD�CY11V; F); pGY9738-Y11A (umuD�C Y11A; Œ).

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recruited to the replication fork and bind DNA but has adefect in polymerase or translesion synthesis function. If thiswere the case, we would expect that additional mutations thatdisrupt recruitment of UmuC to the replication fork wouldsuppress the extreme UV sensitivity caused by the presence ofthe Y11A variant. Binding to the � clamp is critical for UmuCto act in UV-induced mutagenesis due to its role in translesionsynthesis (3, 4). As strains in which the umuC gene has beendeleted have, at most, a very modest sensitivity to UV (30), wepredicted that if the UmuC Y11A variant could not be re-cruited to the replication fork, cells expressing UmuC-Y11Awould no longer be hypersensitive to UV. Two regions ofUmuC have been found to be important for binding to the �clamp. The primary � binding motif, 357QLNLF361 (15), waschanged to ALNAA (referred to as �1), and the other known� binding site, 313LTP315 (4), was changed to AAA (referred toas �2). We previously showed that mutations in the UmuC �1site resulted in a nearly complete loss of UmuC-dependentUV-induced mutagenesis, whereas mutations in the �2 site didnot (4). Furthermore, mutations in either site alone abolishedumuDC-mediated cold sensitivity (4). Each � binding motifwas mutated within the context of the Y11A steric gate variantand a combination of both � binding motif variants togetherwith the steric gate variant was also constructed. These con-structs were then assayed for survival after exposure to UVlight (Fig. 4). Introduction of variants with mutations at �2suppressed the UV-hypersensitive phenotype of Y11A. Thecombination of mutations at both the �1 and �2 sites substan-tially suppressed the UV sensitivity caused by a plasmid ex-pressing UmuC-Y11A. This suggests that UmuC Y11A cannotcause extreme UV hypersensitivity if it is not recruited to the

replication fork by the � clamp. However, even disrupting boththe �1 and �2 sites does not fully suppress the UV hypersen-sitivity caused by UmuC-Y11A, suggesting that other mecha-nisms exist for recruitment of UmuC to damaged DNA, pos-sibly including other interaction sites with the � clamp (26, 63).

Steric gate variants are dominant negative for UV sensitiv-ity. As UmuC steric gate variants cause an unprecedenteddegree of sensitivity to UV light in a umuDC mutant, we nextexamined whether they were dominant negative. We assayedfor UV sensitivity wild-type E. coli strains (AB1157), whichhave a chromosomally encoded copy of the wild-type umuDCoperon, that harbored low-copy-number plasmids expressingthe UmuC F10 and Y11 variants. The UmuC Y11A and Y11Vvariants were dominant negative, causing UV hypersensitivityin the presence of a wild-type copy of UmuC (Fig. 5A). It

FIG. 3. (A) A strain expressing the steric gate variant of UmuCexhibits a growth phenotype similar to wild type. Assays were per-formed with plasmid pGY9738 and derivatives in GW8017: pGY9738(umuD�C; f); pGB2 (empty vector; }); pGY9738-Y11A (umuD�CY11A; Œ). (B) Immunoblot showing steady-state levels of UmuC ex-pressed from variant umuDC plasmids (in GW8017) and from thechromosome in GW2771. Relative UmuC protein levels are indicatedbelow the blot.

FIG. 4. The extreme UV sensitivity is suppressed by mutations inthe � clamp interaction motifs. Assays were performed with plasmidpGY9738 and derivatives in GW8017: pGY9738 (umuD�C; }); pGB2(empty vector; ); pGY9738-Y11A �1&2 (umuD�C Y11A �1&2; �);pGY9738-Y11A �2 (umuD�C Y11A �2; f); pGY9738-Y11A-�1(umuD�C Y11A �1; F); pGY9738-Y11A (umuD�C Y11A; Œ).

FIG. 5. Steric gate variants of UmuC are dominant negative. As-says were performed with plasmid pGY9738 and derivatives in AB1157(umuDC�). (A) pGY9738 (umuD�C; }); pGB2 (empty vector; f);pGY9738-F10V (umuD�C F10V; Œ); pGY9738-Y11V (umuD�CY11V; ); pGY9738-Y11A (umuD�C Y11A; F). (B) pGY9738(umuD�C; }); pGB2 (empty vector; f); pGY9738-Y11A-�1&2(umuD�C Y11A �1&2; Œ); pGY9738-Y11A-�1 (umuD�C Y11A �1;�); pGY9738-Y11A-�2 (umuD�C Y11A �2; ); pGY9738-Y11A(umuD�C Y11A; F).

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should be noted that the UV hypersensitivity observed for theUmuC Y11A variant in the wild-type AB1157 (umuDC�)strain is somewhat attenuated compared to that in GW8017umuDC, which could be due to recombination events thatwould eliminate the variant allele (19, 32). Therefore, thedominant negative phenotype could be underestimated. TheF10A variant exhibited a less dramatic UV-sensitive phenotypebut was still dominant negative. We also assayed strains ex-pressing the UmuC Y11A variant for growth in the context ofAB1157 and found no growth defects (data not shown).

Given our observation that changes to both of the � bindingmotifs of UmuC could partially suppress the UV hypersensi-tivity of umuDC strains harboring plasmids expressing theUmuC Y11A variant (Fig. 4), we investigated whether thesesame changes would suppress the dominant negative pheno-type of the UmuC Y11A variant. We found that alteration ofeither of the known � binding motifs individually, �1 or �2,failed to suppress the dominant negative phenotype of theUmuC Y11A variant plasmid with respect to sensitivity tokilling by UV (Fig. 5B). However, mutation of both �-bindingmotifs in the context of UmuC-Y11A partially suppressed thedominant negative phenotype of UmuC-Y11A (Fig. 5B). Thissuggests that decreased recruitment of UmuC to the replica-tion fork via interactions with the � clamp renders the UmuCY11A variant unable to exert its deleterious effect. Moreover itsuggests that even though the �2 site is dispensable for UmuC-dependent UV-induced mutagenesis (4) it plays a critical rolein recruitment of UmuC to the replication fork.

Steric gate variants are defective in UV-induced mutagene-sis. We assayed the proficiency of these variants for UV-in-duced mutagenesis by determining the argE33arg� reversionfrequency of wild-type (umuDC�) strains harboring theumuD�C plasmids. There is appreciable mutagenesis in theabsence of UV light in strains harboring either pUmuD�C orpY11A plasmids (Fig. 6). This level of mutagenesis is abovethat observed in strains harboring the empty vector and thus isdue to the plasmid-borne umuD�C or the Y11A variant. In-triguingly, upon UV irradiation, the level of mutagenesis in-creases approximately threefold in strains harboring pUmuD�C,whereas in strains harboring the Y11A variant the level ofmutagenesis decreases by almost 2.5-fold. This suggests thatthe UmuC Y11A variant has a specific defect in mutagenesisupon DNA damage, while in the absence of DNA damage theY11A variant is nearly as proficient for mutagenesis as wild-

type UmuC. Strains harboring the p�1 variant showed greatlyreduced, but not a complete loss of, UV-induced mutagenesis,consistent with previous observations (4). Strains harboringplasmids expressing the UmuC-Y11A-�1 variant displayed re-duced mutagenesis in the absence of treatment with UV lightrelative to the Y11A variant and approximately equivalent tothat of the empty vector. The �1 motif is very important forumuC-dependent mutagenesis, whether in the context of wild-type umuC or the Y11A variant, and yet disruption of the motifdoes not completely suppress the UV sensitivity of strainsexpressing UmuC-Y11A. These observations suggest that thedisruption of the �1 motif does not completely prevent recruit-ment of UmuC to DNA but rather prevents formation of acomplex or a conformation required for UV-induced mu-tagenesis. It is also formally possible that the two umuC-dependent phenotypes may be at least partially mechanisti-cally distinct.

UmuC Y11A is dominant negative for sensitivity to otherDNA-damaging agents. Based on our observation of the strik-ing dominant negative phenotype observed with strains ex-pressing the UmuC Y11A variant, we examined its effects in awild-type strain (AB1157) with other DNA-damaging agents,namely, NFZ and MMS. NFZ is predicted to cause mainlyN2-dG adducts (29), whereas MMS is an alkylating agent (20).Strains in which dinB is deleted are sensitive to NFZ (29). TheUmuC Y11A steric gate variant conferred on a wild-type strainextreme sensitivity to both NFZ and MMS, and thus is domi-nant negative (Fig. 7). Therefore, the dominant negative sen-sitivity phenotype of strains expressing UmuC-Y11A is notunique to DNA damage resulting from UV exposure.

The observation that UmuC Y11A conferred NFZ sensitiv-ity made us wonder whether the DinB steric gate variant wouldcause sensitivity to UV light. The steric gate variant of DinBcaused a wild-type E. coli strain to be sensitive to UV light (Fig.7C). Taken together, these observations suggest that whilethere may be specific lesions that are bypassed more efficientlythan others by specific Y family polymerases, either Y familypolymerase may be recruited to bypass any type of DNA dam-age. Moreover, these steric gate variants seem to prevent an-other Y family polymerase from accessing damaged DNA.

UV hypersensitivity of the steric gate variant of UmuC issuppressed by disruption of the � subunit of DNA Pol III.When DNA polymerases encounter lesions in DNA that theycannot replicate, polymerase stalling may occur. There is ge-netic evidence that polymerase stalling is a manifestation of afutile cycle of incorporation of nucleotides opposite the lesionfollowed by exonucleolytic proofreading (46). This process re-generates the primer terminus, which can again be a substratefor DNA polymerase. We considered the formal possibilitythat the UmuC Y11A variant retains polymerase activity butnot translesion synthesis function, as suggested above (Fig. 6),similar to the effects observed with the E. coli DinB steric gatevariant (29). If this were the case, the variant polymerasewould stall at a lesion and may even prevent other repairprocesses from taking place, essentially acting as a competitiveinhibitor. Thus, we investigated the effect that disrupting dnaQ,which encodes the ε subunit of Pol III that is responsible forproofreading (33), would have on the extreme UV sensitivityof cells harboring the Y11A UmuC variant. We compared theUV sensitivity of GW2771, GW2771 spq-2, and GW2771 spq-2

FIG. 6. UV-induced and uninduced arg� revertants of AB1157 har-boring plasmids expressing the umuD�C variants indicated. Black bars,�UV (10 J/m2); white bars, �UV.

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dnaQ903 cells harboring plasmids expressing the Y11A UmuCvariant (Fig. 8A). The spq-2 allele is an antimutator allele ofdnaE which encodes a variant of the DNA Pol III polymerasesubunit DnaE, V832G, and allows cells with a disruption ofdnaQ to grow normally (55). The extreme UV-sensitive dom-inant negative phenotype of the UmuC-Y11A variant was sup-pressed upon disruption of the gene encoding the proofreadingsubunit of Pol III, dnaQ. This may be because a lack of the εsubunit of Pol III either interferes with recruitment of UmuCto the replication fork or abolishes the futile cycle of extensionand excision. This effect may be specific to dnaQ, as strainsharboring variant alleles of other genes whose products areinvolved in DNA replication and repair [dnaN59, recJ, ordnaE(Ts)] and which also harbored pY11A were extremelysensitive to UV light (data not shown).

We exploited our observation that disruption of dnaQ sup-presses the UV sensitivity of the UmuC Y11A variant to de-termine the argE33arg� reversion frequency of dnaQ903 cellsharboring plasmids expressing the Y11A variant. This strain(GW2771 spq-2 dnaQ903) is umuDC�, so the empty vectorgives appreciable UV-induced mutagenesis (Fig. 8B). How-ever, cells harboring plasmids expressing UmuC Y11A exhibiteven lower UV-induced mutagenesis than those harboring theempty vector (Fig. 8B), suggesting that the UmuC Y11A vari-ant suppresses the UV mutagenesis that is due to the chromo-somal copy of umuDC. Therefore, the UmuC Y11A variant isdominant negative with respect to UV mutagenesis.

DISCUSSION

The steric gate residue of DNA polymerases, often Glu, Tyr,or Phe, prevents incorporation of ribonucleotides by stericallyoccluding the 2�-OH moiety (1). The steric gate has beenshown to play this role in preventing rNTP incorporation in theY family of translesion synthesis DNA polymerases (16, 17).We cannot rule out the possibility that expression of UmuCY11A leads to ribonucleotide incorporation, which might beexpected to contribute to sensitivity to DNA-damaging agentsthat is observed, as these treatments increase expression ofUmuC.

It has been observed that the steric gate in E. coli DinB andits orthologs is critical for specific lesion bypass activity (29). Inparticular, the F13V variant of E. coli DinB retains activity asa DNA polymerase on undamaged DNA templates in vitro butlacks translesion synthesis activity (29). We constructed vari-ants of E. coli UmuC with mutations at the steric gate, Y11, as

FIG. 7. Steric gate variants confer sensitivity to the DNA-damagingagents nitrofurazone (A), MMS (B), and UV (C). (A and B) Assayswere performed with plasmid pGY9738 and derivatives in AB1157(umuDC�): pGY9738 (umuD�C; }); pGB2 (empty vector; Œ);pGY9738-Y11A (umuD�C Y11A; f). (C) The DinB steric gate variant(F13V) confers sensitivity to UV light on AB1157 (umuDC�) andderivatives: pYG768 (dinB; f); pWSK30 (empty vector, }); pYG768-F13V (dinB F13V, Œ).

FIG. 8. (A) Disruption of dnaQ, the proofreading subunit, sup-presses the UV hypersensitivity of the UmuC steric gate variant. As-says were performed with plasmid pGY9738-Y11A in GW2771 andderivatives: GW2771 spq-2 (f); GW2771 (}); GW2771 spq-2 dnaQ903(Œ). (B) UV-induced arg� revertants of GW2771 spq-2 dnaQ903 har-boring the plasmids indicated (irradiated at 10 J/m2).

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well as at F10. Strains expressing these UmuC variants dis-played extreme hypersensitivity to UV light. They were alsodominant negative for sensitivity to UV light as well to theDNA-damaging agents NFZ and MMS. In the absence of UVirradiation, strains harboring plasmids expressing wild-typeUmuC or the Y11A variant displayed similar levels of mu-tagenesis. However, upon UV irradiation, mutagenesis in astrain expressing the UmuC-Y11A variant was reduced. Thisobservation suggests that, like DinB (29), the steric gate resi-due of UmuC specifically facilitates translesion synthesis.

The UmuC F10L variant, identified in a screen of forUmuD�C variants that enhance recombination inhibition, es-sentially lacks translesion synthesis activity (57, 59). The Sac-charomyces cerevisiae Pol � F34L variant, at the position anal-ogous to UmuC F10, displayed dramatically reducedtranslesion synthesis activity on a template containing a cis-syncyclobutane pyrimidine dimer while maintaining DNA synthe-sis activity (42). Variants of the S. cerevisiae or human repli-cative DNA polymerase Pol � L868F or L864F, respectively, atthe analogous position to UmuC F10, are able to catalyzebypass of DNA lesions, an activity lacking in the wild-typeenzymes (42). Similarly, S. cerevisiae replicative DNA polymer-ase Pol ε and Pol � variants at the analogous position to UmuCF10 display reduced fidelity (41, 47). Clearly, this position inboth replicative and translesion DNA polymerases is criticalfor the ability to bypass lesions and for replication fidelity.

Strains expressing the UmuC Y11A steric gate variant ex-hibit an unprecedented degree of sensitivity to UV light com-pared to other known umuC alleles. Another example of apoint mutation in UmuC that shows sensitivity to UV light isUmuC A39V, the umuC125 allele (37). This umuDC allele wasidentified in a screen for umuDC variants that failed to conferthe cold sensitivity for growth that is characteristic of elevatedexpression of wild-type umuDC. Cells expressing UmuC A39Vwere sensitive to UV light at 30°C, but not at 43.5°C (37).However, the UV sensitivity observed for strains harboringplasmids expressing UmuC A39V was less than that of UmuCY11A. Intriguingly, the A39 side chain is predicted to be lessthan 4.5 Å from the side chain of Y11 in our homology modelof UmuC (4), raising the possibility that the A39V mutationdisrupts the proper positioning of Y11A in the active site ofUmuC.

The � clamp has been shown to be involved in the recruit-ment of all five of the DNA polymerases in E. coli, making theproteins available for replication as needed. When both of theknown � clamp binding motifs of UmuC (4) are mutated incombination, the UV hypersensitivity of strains expressing theUmuC steric gate variant is suppressed, although not fully. Theobservation that the UmuC Y11A-�1&�2 variant does notfully suppress UV sensitivity in the context of either umuDCor umuDC� strains suggests that, while interactions with the �clamp are necessary for recruitment, there must be additionalmechanisms that facilitate UmuC recruitment to the replica-tion fork. These could include interactions with other compo-nents of the replisome, or with other proteins, such as UmuD2,UmuD�2, or RecA. Additional interactions with the � clampare likely to play a role in the UV sensitivity of strains harbor-ing pY11A-�1&2. In particular, the � clamp loop composed ofresidues 148 to 152 has been shown to be very important for

umuC-dependent mutagenesis (26, 63), although the region ofUmuC that interacts with this loop is unknown.

There is increasing evidence that certain Y family DNApolymerases are specific for a given lesion or a narrow rangeof lesions (29, 34, 48, 65). DinB copies DNA containingN2-furfuryl-dG more efficiently than it copies undamagedDNA, and cells in which dinB is deleted are sensitive to NFZ(29). Pol V (UmuD�2C) copies DNA containing the majorproducts of UV irradiation, thymine-thymine (T-T) cyclobu-tane pyrimidine dimers and T(6-4)T photoproducts and istypically only mutagenic when bypassing the latter (48, 65).We observed that UmuC-Y11A confers sensitivity to NFZon a wild-type strain and that DinB-F13V confers sensitivityto UV light on a wild-type strain. These observations suggesta model of polymerase management in which either E. coliY family polymerase can be recruited to the replication forkupon DNA damage, regardless of the specific lesion that ispresent.

When DNA replication forks encounter lesions, they maystall due to their inability to continue past the lesion (20),leading to uncoupling of leading and lagging strand replication(27, 38, 45). Genetic evidence suggests that the observed DNAreplication stalling may actually be the manifestation of a futilecycle of base insertion opposite the lesion by a DNA polymer-ase followed by exonucleolytic excision of the incorrect base(46). When a lesion bypass DNA polymerase extends a primersufficiently past a lesion, the need for exonucleotytic proof-reading is obviated and replicative DNA polymerases can re-sume replication (21). It seems likely that UmuC Y11A isrecruited to damaged DNA but is then unable to bypass le-sions. Thus, it may prevent other repair processes from takingplace or it may prevent another DNA polymerase from access-ing the replication fork. Since the extreme UV hypersensitivitydue to UmuC Y11A may result from an inability to continuepast a lesion after insertion, we suspected that deletion ordisruption of dnaQ would suppress the UV sensitivity, which itdid. Another possible explanation for the observed suppressionis that the proofreading subunit is responsible for recruitmentof UmuC to replication forks, particularly since a direct phys-ical interaction has been observed between UmuD and ε, thegene product of dnaQ (64). If this were the case, lesions re-sulting from UV irradiation may then be available for directrepair by other pathways or bypass by other DNA polymerases.It has been observed that in strains with a deletion of umuDCand a proofreading-deficient dnaQ allele, thymine-thyminedimer bypass frequencies are not decreased (6). Based onexamination of replication of T-T dimer-containing vectors inthe presence or absence of other DNA polymerases, the T-Tdimer bypass activity in these strains has largely been attrib-uted to Pol III (6).

We report here that E. coli strains expressing UmuC variantsat either the steric gate, Y11, or the adjacent position, F10, arehypersensitive to UV light. The observed degree of UV hyper-sensitivity of the steric gate variants is unprecedented for asingle point mutation of UmuC. The steric gate variant ofUmuC is also strongly dominant negative. Moreover, disrup-tion of the � binding motifs of UmuC suppresses the UVsensitivity, indicating that recruitment to the replication fork isrequired for UmuC Y11A to exert its extremely deleteriouseffect on survival.

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ACKNOWLEDGMENTS

We thank Susan E. Cohen, Daniel F. Jarosz, Sharotka M. Simon,and Kathryn Jones for helpful discussions and technical advice. Wethank Sumati Murli for construction of the GW2771 spq-2 strain andMark Sutton (University at Buffalo, SUNY) and Roger Woodgate(NIH) for strains.

This work was supported by a Camille and Henry Dreyfus Founda-tion New Faculty Award and funding from the Northeastern Univer-sity Office of the Provost to P.J.B. G.C.W. is supported by NIH grantCA21615 from the National Cancer Institute and NIEHS Center grantP30ES02 from the MIT Center for Environmental Health Sciences.G.C.W. is an American Cancer Society Research Professor.

REFERENCES

1. Astatke, M., K. Ng, N. D. Grindley, and C. M. Joyce. 1998. A single sidechain prevents Escherichia coli DNA polymerase I (Klenow fragment) fromincorporating ribonucleotides. Proc. Natl. Acad. Sci. USA 95:3402–3407.

2. Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A.Datsenko, M. Tomita, B. L. Wanner, and H. Mori. 2006. Construction ofEscherichia coli K-12 in-frame, single-gene knockout mutants: the Keio col-lection. Mol. Syst. Biol. 2:2006.0008.

3. Becherel, O. J., R. P. P. Fuchs, and J. Wagner. 2002. Pivotal role of the�-clamp in translesion DNA synthesis and mutagenesis in E. coli cells. DNARepair 1:703–708.

4. Beuning, P. J., D. Sawicka, D. Barsky, and G. C. Walker. 2006. Two pro-cessivity clamp interactions differentially alter the dual activities of UmuC.Mol. Microbiol. 59:460–474.

5. Beuning, P. J., S. M. Simon, V. G. Godoy, D. F. Jarosz, and G. C. Walker.2006. Characterization of Escherichia coli translesion synthesis polymerasesand their accessory factors. Methods Enzymol. 408:318–340.

6. Borden, A., P. I. O’Grady, D. Vandewiele, A. R. Fernandez de Henestrosa,C. W. Lawrence, and R. Woodgate. 2002. Escherichia coli DNA polymeraseIII can replicate efficiently past a T-T cis-syn cyclobutane dimer if DNApolymerase V and the 3� to 5� exonuclease proofreading function encoded bydnaQ are inactivated. J. Bacteriol. 184:2674–2681.

7. Boudsocq, F., H. Ling, W. Yang, and R. Woodgate. 2002. Structure-basedinterpretation of missense mutations in Y-family DNA polymerases andtheir implications for polymerase function and lesion bypass. DNA Repair1:343–358.

8. Bull, H. J., M. J. Lombardo, and S. M. Rosenberg. 2001. Stationary-phasemutation in the bacterial chromosome: recombination protein and DNApolymerase IV dependence. Proc. Natl. Acad. Sci. USA 98:8334–8341.

9. Burckhardt, S. E., R. Woodgate, R. H. Scheuermann, and H. Echols. 1988.UmuD mutagenesis protein of Escherichia coli: overproduction, purification,and cleavage by RecA. Proc. Natl. Acad. Sci. USA 85:1811–1815.

10. Churchward, G., D. Belin, and Y. Nagamine. 1984. A pSC101-derived plas-mid which shows no sequence homology to other commonly used cloningvectors. Gene 31:165–171.

11. Cirz, R. T., J. K. Chin, D. R. Andes, V. de Crecy-Lagard, W. A. Craig, andF. E. Romesberg. 2005. Inhibition of mutation and combating the evolutionof antibiotic resistance. PLoS Biol. 3:e176.

12. Cirz, R. T., M. B. Jones, N. A. Gingles, T. D. Minogue, B. Jarrahi, S. N.Peterson, and F. E. Romesberg. 2007. Complete and SOS-mediated responseof Staphylococcus aureus to the antibiotic ciprofloxacin. J. Bacteriol. 189:531–539.

13. Cirz, R. T., B. M. O’Neill, J. A. Hammond, S. R. Head, and F. E. Romesberg.2006. Defining the Pseudomonas aeruginosa SOS response and its role in theglobal response to the antibiotic ciprofloxacin. J. Bacteriol. 188:7101–7110.

14. Cirz, R. T., and F. E. Romesberg. 2006. Induction and inhibition of cipro-floxacin resistance-conferring mutations in hypermutator bacteria. Antimi-crob. Agents Chemother. 50:220–225.

15. Dalrymple, B. P., K. Kongsuwan, G. Wijffels, N. E. Dixon, and P. A. Jen-nings. 2001. A universal protein-protein interaction motif in the eubacterialDNA replication and repair systems. Proc. Natl. Acad. Sci. USA 98:11627–11632.

16. DeLucia, A. M., S. Chaudhuri, O. Potapova, N. D. Grindley, and C. M. Joyce.2006. The properties of steric gate mutants reveal different constraints withinthe active sites of Y-family and A-family DNA polymerases. J. Biol. Chem.281:27286–27291.

17. DeLucia, A. M., N. D. Grindley, and C. M. Joyce. 2003. An error-pronefamily Y DNA polymerase (DinB homolog from Sulfolobus solfataricus) usesa ‘steric gate’ residue for discrimination against ribonucleotides. NucleicAcids Res. 31:4129–4137.

18. Duzen, J. M., G. C. Walker, and M. D. Sutton. 2004. Identification of specificamino acid residues in the E. coli processivity clamp involved in interactionswith DNA polymerase III, UmuD and UmuD�. DNA Repair 3:301–312.

19. Feil, E. J., and B. G. Spratt. 2001. Recombination and the populationstructures of bacterial pathogens. Annu. Rev. Microbiol. 55:561–590.

20. Friedberg, E. C., G. C. Walker, W. Siede, R. D. Wood, R. A. Schultz, and T.

Ellenberger. 2005. DNA Repair and mutagenesis, 2nd ed. ASM Press, Wash-ington, DC.

21. Fujii, S., and R. P. Fuchs. 2004. Defining the position of the switchesbetween replicative and bypass DNA polymerases. EMBO J. 23:4342–4352.

22. Fujii, S., V. Gasser, and R. P. Fuchs. 2004. The biochemical requirements ofDNA polymerase V-mediated translesion synthesis revisited. J. Mol. Biol.341:405–417.

23. Gao, G., M. Orlova, M. M. Georgiadis, W. A. Hendrickson, and S. P. Goff.1997. Conferring RNA polymerase activity to a DNA polymerase: a singleresidue in reverse transcriptase controls substrate selection. Proc. Natl.Acad. Sci. USA 94:407–411.

24. Gonzalez, M., and R. Woodgate. 2002. The “tale” of UmuD and its role inSOS mutagenesis. Bioessays 24:141–148.

25. Guzzo, A., M. H. Lee, K. Oda, and G. C. Walker. 1996. Analysis of the regionbetween amino acids 30 and 42 of intact UmuD by a monocysteine approach.J. Bacteriol. 178:7295–7303.

26. Heltzel, J. M. H., S. K. Scouten Ponticelli, L. H. Sanders, J. M. Duzen, V.Cody, J. Pace, E. H. Snell, and M. D. Sutton. 2009. Sliding clamp-DNAinteractions are required for viability and contribute to DNA polymerasemanagement in Escherichia coli. J. Mol. Biol. 387:74–91.

27. Higuchi, K., T. Katayama, S. Iwai, M. Hidaka, T. Horiuchi, and H. Maki.2003. Fate of DNA replication fork encountering a single DNA lesion duringoriC plasmid DNA replication in vitro. Genes Cells 8:437–449.

28. Humphrey, W., A. Dalke, and K. Schulten. 1996. VMD: visual moleculardynamics. J. Mol. Graph. 14:33–38.

29. Jarosz, D. F., V. G. Godoy, J. C. DeLaney, J. M. Essigmann, and G. C.Walker. 2006. A single amino acid governs enhanced activity of DinB DNApolymerases on damaged templates. Nature 439:225–228.

30. Kato, T., and Y. Shinoura. 1977. Isolation and characterization of mutants ofEscherichia coli deficient in induction of mutations by ultraviolet light. Mol.Gen. Genet. 156:121–131.

31. 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 instrongly enhancing mutagenesis in the absence of any exogenous treatmentto damage DNA. Proc. Natl. Acad. Sci. USA 94:13792–13797.

32. Kolodner, R., R. A. Fishel, and M. Howard. 1985. Genetic recombination ofbacterial plasmid DNA: effect of RecF pathway mutations on plasmid re-combination in Escherichia coli. J. Bacteriol. 163:1060–1066.

33. Kornberg, A., and T. A. Baker. 1992. DNA replication, 2nd ed. W. H.Freeman & Company, New York, NY.

34. Lee, C. H., S. Chandani, and E. L. Loechler. 2005. Homology modeling offour Y-family lesion-bypass DNA polymerases: the case that E. coli Pol IVand human Pol � are orthologs, and E. coli Pol V and human Pol � areorthologs. J. Mol. Graph. Model. 25:87–102.

35. Lopez de Saro, F. J., R. Georgescu, and M. O’Donnell. 2003. A peptideswitch regulates DNA polymerase processivity. Proc. Natl. Acad. Sci. USA100:14689–14694.

36. Maor-Shoshani, A., and Z. Livneh. 2002. Analysis of the stimulation of DNApolymerase V of Escherichia coli by processivity proteins. Biochemistry 41:14438–14446.

37. Marsh, L., T. Nohmi, S. Hinton, and G. C. Walker. 1991. New mutations incloned Escherichia coli umuDC genes: novel phenotypes of strains carrying aumuC125 plasmid. Mutat. Res. 250:183–197.

38. McInerney, P., and M. O’Donnell. 2004. Functional uncoupling of twinpoymerases. J. Biol. Chem. 279:21543–21551.

39. McKenzie, G. J., P. L. Lee, M. J. Lombardo, P. J. Hastings, and S. M.Rosenberg. 2001. SOS mutator DNA polymerase IV functions in adaptivemutation and not adaptive amplification. Mol. Cell 7:571–579.

40. Neher, S. B., R. T. Sauer, and T. A. Baker. 2003. Distinct peptide signals inthe UmuD and UmuD� subunits of UmuD/D� mediate tethering and sub-strate processing by the ClpXP protease. Proc. Natl. Acad. Sci. USA 100:13219–13224.

41. Nick McIlhinny, S. A., C. M. Stith, P. M. Burgers, and T. A. Kunkel. 2006.Inefficient proofreading and biased error rates during inaccurate DNA syn-thesis by a mutant derivative of Saccharomyces cerevisiae DNA polymerasedelta. J. Biol. Chem. 282:2324–2332.

42. Niimi, A., S. Limsirichaikul, S. Yoshida, S. Iwai, C. Masutani, F. Hanaoka,E. T. Kool, Y. Nishiyama, and M. Suzuki. 2004. Palm mutants in DNApolymerases � and � alter DNA replication fidelity and translesion activity.Mol. Cell. Biol. 24:2734–2746.

43. 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 85:1816–1820.

44. Opperman, T., S. Murli, and G. C. Walker. 1996. The genetic requirementsfor UmuDC-mediated cold sensitivity are distinct from those for SOS mu-tagenesis. J. Bacteriol. 178:4400–4411.

45. Pages, V., and R. P. Fuchs. 2003. Uncoupling of leading- and lagging-strandDNA replication during lesion bypass in vivo. Science 300:1300–1303.

46. Pages, V., R. Janel-Bintz, and R. P. Fuchs. 2005. Pol III proofreading activity

4822 SHURTLEFF ET AL. J. BACTERIOL.

on July 10, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

prevents lesion bypass as evidenced by its molecular signature within E.colicells. J. Mol. Biol. 352:501–509.

47. Pursell, Z. F., I. Isoz, E.-B. Lundstrom, E. Johansson, and T. A. Kunkel.2007. Regulation of B family DNA polymerase fidelity by a conserved activesite residue: characterization of M644W, M644L and M644F mutants ofyeast DNA polymerase ε. Nucleic Acids Res. 35:3076–3086.

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. 274:31763–31766.

49. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Har-bor, NY.

50. Schlacher, K., M. M. Cox, R. Woodgate, and M. F. Goodman. 2006. RecAacts in trans to allow replication of damaged DNA by DNA polymerase V.Nature 442:883–887.

51. Schlacher, K., and M. F. Goodman. 2007. Lessons from 50 years of SOSDNA-damage-induced mutagenesis. Nat. Rev. Mol. Cell Biol. 8:587–594.

52. Schlacher, K., K. Leslie, C. Wyman, R. Woodgate, M. Cox, and M. Goodman.2005. DNA polymerase V and RecA protein, a minimal mutasome. Mol. Cell17:561–572.

53. Schlacher, K., P. Pham, M. M. Cox, and M. F. Goodman. 2006. Roles ofDNA polymerase V and RecA protein in SOS damage-induced mutation.Chem. Rev. 106:406–419.

54. Simmons, L. A., J. J. Foti, S. E. Cohen, and G. C. Walker. July 2008 postingdate. Chapter 5.4.3, The SOS regulatory network. In R. Curtiss III et al.(ed.), EcoSal—Escherichia coli and Salmonella: cellular and molecular biol-ogy. ASM Press, Washington, DC. http://www.ecosal.org.

55. Slater, S. C., M. R. Lifsics, M. O’Donnell, and R. Maurer. 1994. holE, thegene coding for the theta subunit of DNA polymerase III of Escherichia coli:characterization of a holE mutant and comparison with a dnaQ (ε-subunit)mutant. J. Bacteriol. 176:815–821.

56. Slechta, E. S., K. L. Bunny, E. Kugelberg, E. Kofoid, D. I. Andersson, andJ. R. Roth. 2003. Adaptive mutation: general mutagenesis is not a pro-grammed response to stress but results from rare coamplification of dinBwith lac. Proc. Natl. Acad. Sci. USA 100:12847–12852.

57. Sommer, S., O. J. Becherel, G. Coste, A. Bailone, and R. P. Fuchs. 2003.Altered translesion synthesis in E. coli Pol V mutants selected for increasedrecombination inhibition. DNA Repair 2:1361–1369.

58. Sommer, S., F. Boudsocq, R. Devoret, and A. Bailone. 1998. Specific RecAamino acid changes affect RecA-UmuD�C interaction. Mol. Microbiol. 28:281–291.

59. Sommer, S., G. Coste, and A. Bailone. 2000. Specific amino acid changesenhance the anti-recombination activity of the UmuD�C complex. Mol. Mi-crobiol. 35:1443–1453.

60. Sommer, S., J. Knezevic, A. Bailone, and R. Devoret. 1993. Induction of onlyone SOS operon, umuDC, is required for SOS mutagenesis in Escherichiacoli. Mol. Gen. Genet. 239:137–144.

61. Sutton, M., M. Kim, and G. C. Walker. 2001. Genetic and biochemicalcharacterization of a novel umuD mutation: insights into a mechanism forUmuD self-cleavage. J. Bacteriol. 183:347–357.

62. Sutton, M. D., and J. M. Duzen. 2006. Specific amino acid residues in thebeta sliding clamp establish a DNA polymerase usage hierarchy in Esche-richia coli. DNA Repair 5:312–323.

63. Sutton, M. D., J. M. Duzen, and R. W. Maul. 2005. Mutant forms of theEscherichia coli beta sliding clamp that distinguish between its roles in rep-lication and DNA polymerase V-dependent translesion DNA synthesis. Mol.Microbiol. 55:1751–1766.

64. 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 96:12373–12378.

65. Tang, M., P. Pham, X. Shen, J. S. Taylor, M. O’Donnell, R. Woodgate, andM. F. Goodman. 2000. Roles of E. coli DNA polymerases IV and V inlesion-targeted and untargeted SOS mutagenesis. Nature 404:1014–1018.

66. Tang, M., X. Shen, E. G. Frank, M. O’Donnell, R. Woodgate, and M. F.Goodman. 1999. UmuD�2C is an error-prone DNA polymerase, Escherichiacoli pol V. Proc. Natl. Acad. Sci. USA 96:8919–8924.

67. Tompkins, J. D., J. L. Nelson, J. C. Hazel, S. L. Leugers, J. D. Stumpf, andP. L. Foster. 2003. Error-prone polymerase, DNA polymerase IV, is respon-sible for transient hypermutation during adaptive mutation in Escherichiacoli. J. Bacteriol. 185:3469–3472.

68. Vandewiele, D., A. R. Fernandez de Henestrosa, A. R. Timms, B. A. Bridges,and R. Woodgate. 2002. Sequence analysis and phenotypes of five temper-ature sensitive mutator alleles of dnaE, encoding modified alpha-catalyticsubunits of Escherichia coli DNA polymerase III holoenzyme. Mutat. Res.499:85–95.

69. Wagner, J., P. Gruz, S. R. Kim, M. Yamada, K. Matsui, R. P. Fuchs, and T.Nohmi. 1999. The dinB gene encodes a novel E. coli DNA polymerase, DNApol IV, involved in mutagenesis. Mol. Cell 4:281–286.

70. Yang, J., Z. Zhuang, R. M. Roccasecca, M. A. Trakselis, and S. J. Benkovic.2004. The dynamic processivity of the T4 DNA polymerase during replica-tion. Proc. Natl. Acad. Sci. USA 101:8289–8294.

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