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Journal of Bacteriology, August 2009, p. 4815-4823, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.01742-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Steric Gate Variants of UmuC Confer UV Hypersensitivity on Escherichia coli

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

Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115,¹ Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,² Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, Massachusetts 02115³

Received 12 December 2008/ Accepted 18 May 2009

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Y family DNA polymerases are specialized for replication of damaged DNA and represent a major contribution to cellular resistance to DNA lesions. Although the Y family polymerase active sites have fewer contacts with their DNA substrates than replicative DNA polymerases, Y family polymerases appear to exhibit specificity for certain lesions. Thus, mutation of the steric gate residue of Escherichia coli DinB resulted in the specific loss of lesion bypass activity. We constructed variants of E. coli UmuC with mutations of the steric gate residue Y11 and of residue F10 and determined that strains harboring these variants are hypersensitive to UV light. Moreover, these UmuC variants are dominant negative with respect to sensitivity to UV light. The UV hypersensitivity and the dominant negative phenotype are partially suppressed by additional mutations in the known motifs in UmuC responsible for binding to the β processivity clamp, suggesting that the UmuC steric gate variant exerts its effects via access to the replication fork. Strains expressing the UmuC Y11A variant also exhibit decreased UV mutagenesis. Strikingly, disruption of the dnaQ gene encoding the replicative DNA polymerase 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 proofreading subunit in a futile cycle of base insertion/excision with the UmuC steric gate variant.

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All organisms are subject to exogenous and endogenous agents that damage DNA. DNA lesions can lead to polymerase stalling during replication and to mutagenesis, either of which may result in death of the cell (20). Y family DNA polymerases possess a specialized ability to copy damaged DNA in a process known as translesion synthesis and therefore represent a major contribution to DNA damage tolerance. However, Y family polymerases copy undamaged DNA with an elevated error rate and thus may also contribute to mutagenesis (20).

Escherichia coli polymerase V (Pol V), the product of the umuDC genes, and Pol IV, the product of the dinB gene, belong to the Y family of DNA polymerases. These polymerases are characterized by their ability to bypass lesions in DNA and by the low fidelity with which they replicate undamaged DNA, potentially introducing mutations that may be harmful (20). E. coli dinB has thus been implicated in adaptive mutagenesis (8, 39, 56, 67), and both dinB and umuC appear to be important in the development of resistance to antibiotics (11-14, 69). Therefore, the expression and activity of potentially mutagenic Y family DNA polymerases are necessarily tightly regulated. Y family polymerases in E. coli are regulated as part of the SOS response (20), which is initiated by an accumulation of single-stranded DNA (ssDNA) resulting from a cell's efforts to replicate damaged DNA. RecA binds to the ssDNA, forms a RecA:ssDNA nucleoprotein filament, and facilitates the self-cleavage of LexA, the protein responsible for transcriptional repression of the SOS regulon. The decreased intracellular concentration of LexA results in induction of at least 57 genes in E. coli, including genes encoding the Y family DNA polymerases Pol IV and Pol V, which are able to bypass a variety of lesions (20, 53, 54). E. coli Pol IV (DinB) efficiently and accurately copies DNA templates containing N²-furfuryl-dG lesions (29). E. coli Pol V (UmuD'₂C) is able to copy DNA templates containing abasic sites and the products of UV irradiation, including thymine-thymine cyclobutane pyrimidine dimers and thymine-thymine (6-4) photoproducts, and is mutagenic when copying the latter (48, 65).

Once expressed, UmuC activity is regulated by the polymerase manager protein, UmuD. UmuD initially exists as the UmuD₂ dimer; upon interaction with a RecA:ssDNA nucleoprotein filament, UmuD cleaves off its own N-terminal 24 amino acids to form UmuD' (43). The UmuD'₂ form is required for UmuC to be active in translesion synthesis (48, 66).

In addition to the transcriptional and posttranslational regulation of UmuC, another layer of regulation exists in that the polymerase must gain access to the damaged DNA for lesion bypass to occur. While much remains to be understood about the recruitment of polymerases in E. coli, protein-protein interactions, particularly with subunits of the replisome and with RecA, are important for regulating access of UmuC to the replication fork (3, 4, 22, 26, 36, 50-52, 63). Whereas only a few direct physical interactions have been shown between UmuC and these proteins, there are numerous demonstrated interactions between the manager protein UmuD and/or UmuD' and other cellular proteins (9, 24, 40, 61, 64).

Interaction with the β clamp is thought to be integral to the coordination of UmuC function (3, 4, 18, 62-64). The β processivity clamp is a ring-shaped dimer that has one "hydrophobic patch" per monomer to which the polymerases in E. coli bind. All five E. coli DNA polymerases have been shown to bind to the β clamp, four of which bind via a QL[S/D]LF motif (15, 35). The β clamp has been shown to increase the processivity of Pol III and Pol IV substantially, but the increase in processivity conferred on Pol V by the β clamp is relatively modest (22, 36). Recent studies of bacteriophage T4 DNA replication suggest a dynamic interaction in phage T4 in which polymerases are rapidly exchanged at the replication fork, a process facilitated by interactions with the processivity clamp (70). Moreover, it has been suggested that polymerase stalling may be due to a futile cycle of base insertion and exonucleolytic proofreading at sites of DNA damage (46). Dynamic interactions at the replication fork could provide an opportunity for alternate polymerases to be employed in lesion bypass and allow DNA replication to continue after damage (21).

DNA polymerases possess a steric gate residue near the active site responsible for the ability of the polymerase to prevent the incorporation of ribonucleotides in DNA by blocking the 2'-OH of incoming ribonucleotides (1, 23). The residue is conserved in Y family polymerases as either Tyr or Phe (Fig. 1) (7, 17). In the case of E. coli DinB, mutation of the steric gate residue (F13V) resulted in an increase of rNTP incorporation from a frequency of <10^–5 to 10^–3 (29). In the DinB subfamily of Y family polymerases, including E. coli DinB and mammalian Pol , the steric gate has been shown to be critical for the specific bypass of DNA lesions (29). Mutation of the steric gate residue (F13V) in E. coli DinB eliminated bypass of N²-furfuryl-dG lesions but did not affect DNA polymerase activity on undamaged DNA (29). We show here that a single substitution in the steric gate residue (Y11) of E. coli UmuC renders cells hypersensitive to UV light and is dominant negative with respect to sensitivity to UV and other DNA damaging agents. The dominant negative phenotype is suppressed by additional mutations within UmuC that disrupt binding to the β clamp or by disruption of dnaQ, the proofreading subunit of the replicative DNA polymerase. We also show that UmuC-Y11A confers decreased UV mutability on cells that harbor this variant.

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FIG. 1. The steric gate residue of UmuC is predicted to approach the incoming nucleotide. (Top) Homology model of UmuC (4) showing the template (green), primer and incoming nucleotide (blue), and residues F10 and steric gate Y11 (red). The backbone of UmuC is shown in yellow. The illustration was prepared using VMD (28). (Bottom) Amino acid sequences of representative Y family polymerases showing 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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Strains, plasmids, and general techniques. The strains and plasmids used in this study are listed in Table 1. The o_1^c mutation present in the operator sequence of both pGY9738 and pGY9739 is a single GA mutation in the SOS consensus box and results in twofold-higher expression levels of UmuD compared to plasmids harboring the o⁺ operator sequence (60). However, expression levels of UmuD from o₁^c plasmids in LexA⁺ strains are lower than in strains that lack LexA; thus, the o₁^c mutation leads to only partial derepression (60). Steric gate (Y11A, Y11V, and F10V) and β clamp interaction mutants (³¹³LTP³¹⁵ to AAA and ³⁵⁷QLNLF³⁶¹ to ALNAA) (4) of UmuC were constructed using the QuikChange site-directed mutagenesis kit (Stratagene). The mutations were confirmed by DNA sequence analysis (Massachusetts General Hospital Core Facility, Cambridge, MA, and MIT Biopolymers Laboratory, Cambridge, MA). Competent cells were prepared using the CaCl₂ method (49). Transformations and immunoblotting were performed as described previously (5). Expression levels of UmuC were quantified using ImageQuant (GE Healthcare) by normalizing to the cross-reacting band.

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TABLE 1. Strains and plasmids

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

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

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

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FIG. 8. (A) Disruption of dnaQ, the proofreading subunit, suppresses the UV hypersensitivity of the UmuC steric gate variant. Assays were performed with plasmid pGY9738-Y11A in GW2771 and derivatives: GW2771 spq-2 (); GW2771 (); GW2771 spq-2 dnaQ903 (). (B) UV-induced arg+ revertants of GW2771 spq-2 dnaQ903 harboring the plasmids indicated (irradiated at 10 J/m2).

Growth assay. Selected colonies from fresh transformations were grown overnight in LB supplemented with spectinomycin (60 µg/ml). Dilutions of the overnight cultures were made to an OD₆₀₀ of 0.05 and were grown at 37°C with shaking. From 0 h to 3 h samples were taken every half hour and the concentration assessed by measuring the OD₆₀₀ and plating on 1.2% LB agar plates supplemented with spectinomycin. From 4 h to 8 h samples were taken every hour and the concentration was determined by measuring the OD₆₀₀.

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Steric gate variants of UmuC render E. coli hypersensitive to UV light. The steric gate residue of DNA polymerases plays a critical role in preventing incorporation of ribonucleotides into DNA (1, 23). In the case of E. coli DinB, the F13V steric gate variant retained the ability to discriminate against ribonucleotides, albeit less proficiently than wild-type DinB (29). However, it appears that, at least for E. coli DinB and its mammalian and archaeal ortholog, an additional role of the steric gate residue is to facilitate lesion bypass (29). In order to assess the role in DNA damage tolerance of the steric gate in E. coli UmuC, we made alanine and valine mutations in UmuC at Y11, as this residue is predicted to be the steric gate residue based on sequence alignments (Fig. 1) (7). We also constructed UmuC F10V, as the position immediately N-terminal to the steric gate is a conserved Phe residue that in the cases of E. coli UmuC (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 expresses umuD'C, rather than umuDC, in order to eliminate any possible confounding effects due to changes in UmuD cleavage efficiency (58). GW8017 (AB1157 umuDC) strains harboring low-copy-number plasmids bearing UmuC variants were assayed for sensitivity to UV light. To our surprise, strains harboring plasmids expressing the Y11A variant of UmuC not only failed to complement the umuDC strain for UV-induced mutagenesis but dramatically sensitized the cells to killing by UV light (Fig. 2). Strikingly, the UmuC Y11A variant renders umuDC cells so sensitive to UV light that extreme killing is observed at a dose of only 5 J/m² (Fig. 2). A similar UV-sensitive phenotype was observed with the Y11A variant assayed in the context of pGY9739 (umuDC) (data not shown). The Y11V variant also caused UV hypersensitivity, whereas the F10V variant caused more modest, but still striking, sensitivity (Fig. 2) comparable to that observed with a catalytically inactive UmuC variant (C104) (Fig. 2). As has been noted previously, loss of umuC has only a modest effect on UV sensitivity (20, 30), but here we found that a single point mutation in UmuC causes an increase in UV sensitivity of several orders of magnitude. To the best of our knowledge, this is the first example of a point mutation in UmuC that renders cells sensitive to UV light to this extent.

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FIG. 2. The steric gate Y11 and F10 variants in UmuC cause UV hypersensitivity. Assays were performed with the pGY9738 plasmid and the following derivatives in GW8017: pGY9738 (umuD'C; ); pGB2 (empty vector; x); pGY9738-C104 (cat-) (umuD'C C104; –); pGY9738-F10V (umuD'C F10V; ); pGY9738-Y11V (umuD'C Y11V; •); pGY9738-Y11A (umuD'C Y11A; ).

The UmuC steric gate variant could confer UV hypersensitivity either via a growth defect of the strain or by a specific defect in polymerase or translesion synthesis activity. We assayed for growth the umuDC strain harboring a plasmid encoding the UmuC Y11A steric gate variant. The growth rates of the strains carrying plasmids encoding the UmuC steric gate variant were indistinguishable from the wild type in the absence of UV irradiation (Fig. 3A). We also confirmed that the steady-state protein levels did not differ substantially between the different constructs, as levels of UmuC varied approximately twofold among the strains that harbor plasmids (Fig. 3B). Approximately threefold more UmuC is expressed from pGY9738 (in GW8017) than from the chromosome in GW2771. Under these conditions, we did not detect UmuC in any of these samples in the absence of UV induction.

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FIG. 3. (A) A strain expressing the steric gate variant of UmuC exhibits a growth phenotype similar to wild type. Assays were performed with plasmid pGY9738 and derivatives in GW8017: pGY9738 (umuD'C; ); pGB2 (empty vector; ); pGY9738-Y11A (umuD'C Y11A; ). (B) Immunoblot showing steady-state levels of UmuC expressed from variant umuDC plasmids (in GW8017) and from the chromosome in GW2771. Relative UmuC protein levels are indicated below the blot.

β clamp interaction motifs modulate the UV hypersensitivity of the steric gate variant. We hypothesized that the UmuC Y11A variant causes UV hypersensitivity because it can still be recruited to the replication fork and bind DNA but has a defect in polymerase or translesion synthesis function. If this were the case, we would expect that additional mutations that disrupt recruitment of UmuC to the replication fork would suppress the extreme UV sensitivity caused by the presence of the Y11A variant. Binding to the β clamp is critical for UmuC to act in UV-induced mutagenesis due to its role in translesion synthesis (3, 4). As strains in which the umuC gene has been deleted have, at most, a very modest sensitivity to UV (30), we predicted that if the UmuC Y11A variant could not be recruited to the replication fork, cells expressing UmuC-Y11A would no longer be hypersensitive to UV. Two regions of UmuC have been found to be important for binding to the β clamp. The primary β binding motif, ³⁵⁷QLNLF³⁶¹ (15), was changed to ALNAA (referred to as β1), and the other known β binding site, ³¹³LTP³¹⁵ (4), was changed to AAA (referred to as β2). We previously showed that mutations in the UmuC β1 site resulted in a nearly complete loss of UmuC-dependent UV-induced mutagenesis, whereas mutations in the β2 site did not (4). Furthermore, mutations in either site alone abolished umuDC-mediated cold sensitivity (4). Each β binding motif was mutated within the context of the Y11A steric gate variant and a combination of both β binding motif variants together with the steric gate variant was also constructed. These constructs were then assayed for survival after exposure to UV light (Fig. 4). Introduction of variants with mutations at β2 suppressed the UV-hypersensitive phenotype of Y11A. The combination of mutations at both the β1 and β2 sites substantially suppressed the UV sensitivity caused by a plasmid expressing UmuC-Y11A. This suggests that UmuC Y11A cannot cause extreme UV hypersensitivity if it is not recruited to the replication fork by the β clamp. However, even disrupting both the β1 and β2 sites does not fully suppress the UV hypersensitivity caused by UmuC-Y11A, suggesting that other mechanisms exist for recruitment of UmuC to damaged DNA, possibly including other interaction sites with the β clamp (26, 63).

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FIG. 4. The extreme UV sensitivity is suppressed by mutations in the β clamp interaction motifs. Assays were performed with plasmid pGY9738 and derivatives in GW8017: pGY9738 (umuD'C; ); pGB2 (empty vector; x); pGY9738-Y11A β1&2 (umuD'C Y11A β1&2; –); pGY9738-Y11A β2 (umuD'C Y11A β2; ); pGY9738-Y11A-β1 (umuD'C Y11A β1; •); pGY9738-Y11A (umuD'C Y11A; ).

Steric gate variants are dominant negative for UV sensitivity. As UmuC steric gate variants cause an unprecedented degree of sensitivity to UV light in a umuDC mutant, we next examined whether they were dominant negative. We assayed for UV sensitivity wild-type E. coli strains (AB1157), which have a chromosomally encoded copy of the wild-type umuDC operon, that harbored low-copy-number plasmids expressing the UmuC F10 and Y11 variants. The UmuC Y11A and Y11V variants were dominant negative, causing UV hypersensitivity in the presence of a wild-type copy of UmuC (Fig. 5A). It should be noted that the UV hypersensitivity observed for the UmuC Y11A variant in the wild-type AB1157 (umuDC⁺) strain is somewhat attenuated compared to that in GW8017 umuDC, which could be due to recombination events that would eliminate the variant allele (19, 32). Therefore, the dominant negative phenotype could be underestimated. The F10A variant exhibited a less dramatic UV-sensitive phenotype but was still dominant negative. We also assayed strains expressing the UmuC Y11A variant for growth in the context of AB1157 and found no growth defects (data not shown).

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FIG. 5. Steric gate variants of UmuC are dominant negative. Assays were performed with plasmid pGY9738 and derivatives in AB1157 (umuDC+). (A) pGY9738 (umuD'C; ); pGB2 (empty vector; ); pGY9738-F10V (umuD'C F10V; ); pGY9738-Y11V (umuD'C Y11V; x); pGY9738-Y11A (umuD'C Y11A; •). (B) pGY9738 (umuD'C; ); pGB2 (empty vector; ); pGY9738-Y11A-β1&2 (umuD'C Y11A β1&2; ); pGY9738-Y11A-β1 (umuD'C Y11A β1; –); pGY9738-Y11A-β2 (umuD'C Y11A β2; x); pGY9738-Y11A (umuD'C Y11A; •).

Given our observation that changes to both of the β binding motifs of UmuC could partially suppress the UV hypersensitivity of umuDC strains harboring plasmids expressing the UmuC Y11A variant (Fig. 4), we investigated whether these same changes would suppress the dominant negative phenotype of the UmuC Y11A variant. We found that alteration of either of the known β binding motifs individually, β1 or β2, failed to suppress the dominant negative phenotype of the UmuC Y11A variant plasmid with respect to sensitivity to killing by UV (Fig. 5B). However, mutation of both β-binding motifs in the context of UmuC-Y11A partially suppressed the dominant negative phenotype of UmuC-Y11A (Fig. 5B). This suggests that decreased recruitment of UmuC to the replication fork via interactions with the β clamp renders the UmuC Y11A variant unable to exert its deleterious effect. Moreover it suggests that even though the β2 site is dispensable for UmuC-dependent UV-induced mutagenesis (4) it plays a critical role in recruitment of UmuC to the replication fork.

Steric gate variants are defective in UV-induced mutagenesis. We assayed the proficiency of these variants for UV-induced mutagenesis by determining the argE3arg⁺ reversion frequency of wild-type (umuDC⁺) strains harboring the umuD'C plasmids. There is appreciable mutagenesis in the absence of UV light in strains harboring either pUmuD'C or pY11A plasmids (Fig. 6). This level of mutagenesis is above that observed in strains harboring the empty vector and thus is due to the plasmid-borne umuD'C or the Y11A variant. Intriguingly, upon UV irradiation, the level of mutagenesis increases approximately threefold in strains harboring pUmuD'C, whereas in strains harboring the Y11A variant the level of mutagenesis decreases by almost 2.5-fold. This suggests that the UmuC Y11A variant has a specific defect in mutagenesis upon DNA damage, while in the absence of DNA damage the Y11A variant is nearly as proficient for mutagenesis as wild-type UmuC. Strains harboring the pβ1 variant showed greatly reduced, but not a complete loss of, UV-induced mutagenesis, consistent with previous observations (4). Strains harboring plasmids expressing the UmuC-Y11A-β1 variant displayed reduced mutagenesis in the absence of treatment with UV light relative to the Y11A variant and approximately equivalent to that of the empty vector. The β1 motif is very important for umuC-dependent mutagenesis, whether in the context of wild-type umuC or the Y11A variant, and yet disruption of the motif does not completely suppress the UV sensitivity of strains expressing UmuC-Y11A. These observations suggest that the disruption of the β1 motif does not completely prevent recruitment of UmuC to DNA but rather prevents formation of a complex or a conformation required for UV-induced mutagenesis. It is also formally possible that the two umuC-dependent phenotypes may be at least partially mechanistically distinct.

UmuC Y11A is dominant negative for sensitivity to other DNA-damaging agents. Based on our observation of the striking dominant negative phenotype observed with strains expressing the UmuC Y11A variant, we examined its effects in a wild-type strain (AB1157) with other DNA-damaging agents, namely, NFZ and MMS. NFZ is predicted to cause mainly N²-dG adducts (29), whereas MMS is an alkylating agent (20). Strains in which dinB is deleted are sensitive to NFZ (29). The UmuC Y11A steric gate variant conferred on a wild-type strain extreme sensitivity to both NFZ and MMS, and thus is dominant negative (Fig. 7). Therefore, the dominant negative sensitivity phenotype of strains expressing UmuC-Y11A is not unique to DNA damage resulting from UV exposure.

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FIG. 7. Steric gate variants confer sensitivity to the DNA-damaging agents nitrofurazone (A), MMS (B), and UV (C). (A and B) Assays were performed with plasmid pGY9738 and derivatives in AB1157 (umuDC+): pGY9738 (umuD'C; ); pGB2 (empty vector; ); pGY9738-Y11A (umuD'C Y11A; ). (C) The DinB steric gate variant (F13V) confers sensitivity to UV light on AB1157 (umuDC+) and derivatives: pYG768 (dinB; ); pWSK30 (empty vector, ); pYG768-F13V (dinB F13V, ).

The observation that UmuC Y11A conferred NFZ sensitivity made us wonder whether the DinB steric gate variant would cause sensitivity to UV light. The steric gate variant of DinB caused a wild-type E. coli strain to be sensitive to UV light (Fig. 7C). Taken together, these observations suggest that while there may be specific lesions that are bypassed more efficiently than others by specific Y family polymerases, either Y family polymerase may be recruited to bypass any type of DNA damage. Moreover, these steric gate variants seem to prevent another Y family polymerase from accessing damaged DNA.

UV hypersensitivity of the steric gate variant of UmuC is suppressed by disruption of the subunit of DNA Pol III. When DNA polymerases encounter lesions in DNA that they cannot replicate, polymerase stalling may occur. There is genetic evidence that polymerase stalling is a manifestation of a futile cycle of incorporation of nucleotides opposite the lesion followed by exonucleolytic proofreading (46). This process regenerates the primer terminus, which can again be a substrate for DNA polymerase. We considered the formal possibility that the UmuC Y11A variant retains polymerase activity but not translesion synthesis function, as suggested above (Fig. 6), similar to the effects observed with the E. coli DinB steric gate variant (29). If this were the case, the variant polymerase would stall at a lesion and may even prevent other repair processes from taking place, essentially acting as a competitive inhibitor. Thus, we investigated the effect that disrupting dnaQ, which encodes the subunit of Pol III that is responsible for proofreading (33), would have on the extreme UV sensitivity of cells harboring the Y11A UmuC variant. We compared the UV sensitivity of GW2771, GW2771 spq-2, and GW2771 spq-2 dnaQ903 cells harboring plasmids expressing the Y11A UmuC variant (Fig. 8A). The spq-2 allele is an antimutator allele of dnaE which encodes a variant of the DNA Pol III polymerase subunit DnaE, V832G, and allows cells with a disruption of dnaQ to grow normally (55). The extreme UV-sensitive dominant negative phenotype of the UmuC-Y11A variant was suppressed upon disruption of the gene encoding the proofreading subunit of Pol III, dnaQ. This may be because a lack of the subunit of Pol III either interferes with recruitment of UmuC to the replication fork or abolishes the futile cycle of extension and excision. This effect may be specific to dnaQ, as strains harboring variant alleles of other genes whose products are involved in DNA replication and repair [dnaN59, recJ, or dnaE(Ts)] and which also harbored pY11A were extremely sensitive to UV light (data not shown).

We exploited our observation that disruption of dnaQ suppresses the UV sensitivity of the UmuC Y11A variant to determine the argE3arg⁺ reversion frequency of dnaQ903 cells harboring plasmids expressing the Y11A variant. This strain (GW2771 spq-2 dnaQ903) is umuDC⁺, so the empty vector gives appreciable UV-induced mutagenesis (Fig. 8B). However, cells harboring plasmids expressing UmuC Y11A exhibit even lower UV-induced mutagenesis than those harboring the empty vector (Fig. 8B), suggesting that the UmuC Y11A variant suppresses the UV mutagenesis that is due to the chromosomal copy of umuDC. Therefore, the UmuC Y11A variant is dominant negative with respect to UV mutagenesis.

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The steric gate residue of DNA polymerases, often Glu, Tyr, or Phe, prevents incorporation of ribonucleotides by sterically occluding the 2'-OH moiety (1). The steric gate has been shown to play this role in preventing rNTP incorporation in the Y family of translesion synthesis DNA polymerases (16, 17). We cannot rule out the possibility that expression of UmuC Y11A leads to ribonucleotide incorporation, which might be expected to contribute to sensitivity to DNA-damaging agents that is observed, as these treatments increase expression of UmuC.

It has been observed that the steric gate in E. coli DinB and its orthologs is critical for specific lesion bypass activity (29). In particular, the F13V variant of E. coli DinB retains activity as a DNA polymerase on undamaged DNA templates in vitro but lacks translesion synthesis activity (29). We constructed variants of E. coli UmuC with mutations at the steric gate, Y11, as well as at F10. Strains expressing these UmuC variants displayed extreme hypersensitivity to UV light. They were also dominant negative for sensitivity to UV light as well to the DNA-damaging agents NFZ and MMS. In the absence of UV irradiation, strains harboring plasmids expressing wild-type UmuC or the Y11A variant displayed similar levels of mutagenesis. However, upon UV irradiation, mutagenesis in a strain expressing the UmuC-Y11A variant was reduced. This observation suggests that, like DinB (29), the steric gate residue of UmuC specifically facilitates translesion synthesis.

The UmuC F10L variant, identified in a screen of for UmuD'C variants that enhance recombination inhibition, essentially lacks translesion synthesis activity (57, 59). The Saccharomyces cerevisiae Pol F34L variant, at the position analogous to UmuC F10, displayed dramatically reduced translesion synthesis activity on a template containing a cis-syn cyclobutane pyrimidine dimer while maintaining DNA synthesis activity (42). Variants of the S. cerevisiae or human replicative DNA polymerase Pol L868F or L864F, respectively, at the analogous position to UmuC F10, are able to catalyze bypass of DNA lesions, an activity lacking in the wild-type enzymes (42). Similarly, S. cerevisiae replicative DNA polymerase Pol and Pol variants at the analogous position to UmuC F10 display reduced fidelity (41, 47). Clearly, this position in both replicative and translesion DNA polymerases is critical for the ability to bypass lesions and for replication fidelity.

Strains expressing the UmuC Y11A steric gate variant exhibit an unprecedented degree of sensitivity to UV light compared to other known umuC alleles. Another example of a point mutation in UmuC that shows sensitivity to UV light is UmuC A39V, the umuC125 allele (37). This umuDC allele was identified in a screen for umuDC variants that failed to confer the cold sensitivity for growth that is characteristic of elevated expression of wild-type umuDC. Cells expressing UmuC A39V were sensitive to UV light at 30°C, but not at 43.5°C (37). However, the UV sensitivity observed for strains harboring plasmids expressing UmuC A39V was less than that of UmuC Y11A. Intriguingly, the A39 side chain is predicted to be less than 4.5 Å from the side chain of Y11 in our homology model of UmuC (4), raising the possibility that the A39V mutation disrupts the proper positioning of Y11A in the active site of UmuC.

The β clamp has been shown to be involved in the recruitment of all five of the DNA polymerases in E. coli, making the proteins available for replication as needed. When both of the known β clamp binding motifs of UmuC (4) are mutated in combination, the UV hypersensitivity of strains expressing the UmuC steric gate variant is suppressed, although not fully. The observation that the UmuC Y11A-β1&β2 variant does not fully suppress UV sensitivity in the context of either umuDC or umuDC⁺ strains suggests that, while interactions with the β clamp are necessary for recruitment, there must be additional mechanisms that facilitate UmuC recruitment to the replication fork. These could include interactions with other components of the replisome, or with other proteins, such as UmuD₂, UmuD'₂, or RecA. Additional interactions with the β clamp are likely to play a role in the UV sensitivity of strains harboring pY11A-β1&2. In particular, the β clamp loop composed of residues 148 to 152 has been shown to be very important for umuC-dependent mutagenesis (26, 63), although the region of UmuC that interacts with this loop is unknown.

There is increasing evidence that certain Y family DNA polymerases are specific for a given lesion or a narrow range of lesions (29, 34, 48, 65). DinB copies DNA containing N²-furfuryl-dG more efficiently than it copies undamaged DNA, and cells in which dinB is deleted are sensitive to NFZ (29). Pol V (UmuD'₂C) copies DNA containing the major products of UV irradiation, thymine-thymine (T-T) cyclobutane pyrimidine dimers and T(6-4)T photoproducts and is typically only mutagenic when bypassing the latter (48, 65). We observed that UmuC-Y11A confers sensitivity to NFZ on a wild-type strain and that DinB-F13V confers sensitivity to UV light on a wild-type strain. These observations suggest a model of polymerase management in which either E. coli Y family polymerase can be recruited to the replication fork upon DNA damage, regardless of the specific lesion that is present.

When DNA replication forks encounter lesions, they may stall 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 DNA replication stalling may actually be the manifestation of a futile cycle of base insertion opposite the lesion by a DNA polymerase followed by exonucleolytic excision of the incorrect base (46). When a lesion bypass DNA polymerase extends a primer sufficiently past a lesion, the need for exonucleotytic proofreading is obviated and replicative DNA polymerases can resume replication (21). It seems likely that UmuC Y11A is recruited to damaged DNA but is then unable to bypass lesions. Thus, it may prevent other repair processes from taking place or it may prevent another DNA polymerase from accessing the replication fork. Since the extreme UV hypersensitivity due to UmuC Y11A may result from an inability to continue past a lesion after insertion, we suspected that deletion or disruption of dnaQ would suppress the UV sensitivity, which it did. Another possible explanation for the observed suppression is that the proofreading subunit is responsible for recruitment of UmuC to replication forks, particularly since a direct physical interaction has been observed between UmuD and , the gene product of dnaQ (64). If this were the case, lesions resulting from UV irradiation may then be available for direct repair by other pathways or bypass by other DNA polymerases. It has been observed that in strains with a deletion of umuDC and a proofreading-deficient dnaQ allele, thymine-thymine dimer bypass frequencies are not decreased (6). Based on examination of replication of T-T dimer-containing vectors in the presence or absence of other DNA polymerases, the T-T dimer bypass activity in these strains has largely been attributed to Pol III (6).

We report here that E. coli strains expressing UmuC variants at either the steric gate, Y11, or the adjacent position, F10, are hypersensitive to UV light. The observed degree of UV hypersensitivity of the steric gate variants is unprecedented for a single point mutation of UmuC. The steric gate variant of UmuC is also strongly dominant negative. Moreover, disruption of the β binding motifs of UmuC suppresses the UV sensitivity, indicating that recruitment to the replication fork is required for UmuC Y11A to exert its extremely deleterious effect on survival.

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

This work was supported by a Camille and Henry Dreyfus Foundation New Faculty Award and funding from the Northeastern University Office of the Provost to P.J.B. G.C.W. is supported by NIH grant CA21615 from the National Cancer Institute and NIEHS Center grant P30ES02 from the MIT Center for Environmental Health Sciences. G.C.W. is an American Cancer Society Research Professor.

 
* Corresponding author. Mailing address: Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, 102 Hurtig Hall, Boston, MA 02115. Phone: (617) 373-2865. Fax: (617) 373-8795. E-mail: beuning{at}neu.edu

Published ahead of print on 29 May 2008.

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Journal of Bacteriology, August 2009, p. 4815-4823, Vol. 191, No. 15
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