Altered translesion synthesis in E. coli Pol V mutants selected for increased recombination inhibition

DNA Repair 2 (2003) 1361–1369

Altered translesion synthesis in E. coli Pol V mutants selected for increased recombination inhibition Suzanne Sommer a,∗ , Olivier J. Becherel b,1 , Geneviève Coste a , Adriana Bailone a , Robert P.P. Fuchs b a

Institut de Génétique et Microbiologie, Bˆat. 409, Université Paris-Sud, F-91405 Orsay, France Cancérogénèse et Mutagenèse Moléculaire et Structurale, UPR 9003 CNRS, UPR Conventionnée avec l’Université Louis Pasteur de Strasbourg , ESBS, Bld Sébastien Brant, F-67400 Illkirch, France b

Received 25 June 2003; accepted 7 August 2003

Abstract Replication of damaged DNA, also termed as translesion synthesis (TLS), involves specialized DNA polymerases that bypass DNA lesions. In Escherichia coli, although TLS can involve one or a combination of DNA polymerases depending on the nature of the lesion, it generally requires the Pol V DNA polymerase (formed by two SOS proteins, UmuD and UmuC) and the RecA protein. In addition to being an essential component of translesion DNA synthesis, Pol V is also an antagonist of RecA-mediated recombination. We have recently isolated umuD and umuC mutants on the basis of their increased capacity to inhibit homologous recombination. Despite the capacity of these mutants to form a Pol V complex and to interact with the RecA polymer, most of them exhibit a defect in TLS. Here, we further characterize the TLS activity of these Pol V mutants in vivo by measuring the extent of error-free and mutagenic bypass at a single (6-4)TT lesion located in double stranded plasmid DNA. TLS is markedly decreased in most Pol V mutants that we analyzed (8/9) with the exception of one UmuC mutant (F287L) that exhibits wild-type bypass activity. Somewhat unexpectedly, Pol V mutants that are partially deficient in TLS are more severely affected in mutagenic bypass compared to error-free synthesis. The defect in bypass activity of the Pol V mutant polymerases is discussed in light of the location of the respective mutations in the 3D structure of UmuD and the DinB/UmuC homologous protein Dpo4 of Sulfolobus solfataricus. © 2003 Elsevier B.V. All rights reserved. Keywords: SOS mutagenesis; E. coli Pol V DNA polymerase; Translesion synthesis; UV-induced base substitution mutagenesis; Bypass polymerase

1. Introduction

∗ Corresponding author. Tel.: +33-1-69-15-46-14; fax: +33-1-69-15-78-08. E-mail address: [email protected] (S. Sommer). 1 Present address: Radiation Biology and Oncology Laboratory, Queensland Institute of Medical Research (QIMR), The Bancroft Centre, P.O. Royal Brisbane Hospital, Brisbane 4029, Qld, Australia.

Despite multiple and efficient DNA repair processes that have evolved to cope with a large variety of DNA damage events, some lesions escape repair and, as a consequence, represent a possible threat for the DNA replication process. Replication of damaged DNA molecules is achieved via two major strategies: (i) an error-free process called damage avoidance (DA), that involves homologous recombination using

1568-7864/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2003.08.008

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the information contained in the locally-undamaged complementary strand to complete replication and/or to fill in the gap generated by a stalled DNA replication fork at a site of DNA damage [1–4] and (ii) translesion synthesis (TLS), a potentially mutagenic process, which uses the damaged strand as a template for replication [5–7] (reviewed by [8]). TLS involves specialized DNA polymerases that bypass DNA lesions (reviewed by [8–15]). In Escherichia coli, although TLS can involve one or a combination of DNA polymerases depending on the nature of the lesion, it generally requires the UmuD2 C complex and the RecA protein [16–19]. The UmuD2 C complex was shown to possess an intrinsic DNA polymerase activity [20,21] and was subsequently identified as the fifth DNA polymerase in E. coli and thus named DNA Polymerase V (Pol V). Pol V is a member of the newly discovered Y-family of DNA polymerases that are specifically involved in TLS [14]. By forming a helical polymer around single-stranded DNA, RecA protein facilitates cleavage of LexA, the SOS repressor, and cleavage of UmuD, the precursor of the mutagenesis protein UmuD , thus allowing the expression of UmuD, UmuC and the formation of the UmuD2 C complex (for reviews, see [22–25]). In addition to its coprotease function, the RecA polymer also plays an essential and direct role in SOS mutagenesis, as evidenced by in vivo [26–29] and in vitro experiments [20,21,30–32]. In addition to being an essential component of translesion DNA synthesis, Pol V is also, when it is expressed constitutively in the cells, an antagonist of RecA-mediated homologous recombination [33,34]. Similarly, purified Pol V has also been found to inhibit RecA-promoted homologous pairing and DNA strand exchange in vitro [4,35]. Inhibition of homologous recombination is not observed if one of the two proteins of the Pol V complex is missing [4,33]. Using a genetic screen, we recently isolated umuD and umuC mutations that enhance recombination inhibition in bacteria overproducing Pol V polymerase [36]. The capacity of the mutants to strongly inhibit homologous recombination suggests that they form a UmuD2 C complex (Pol V) interacting with a RecA polymer [4,35,36]. However, several of these Pol V mutants were unable to promote efficient SOS mutagenesis, as shown by the frequency of lacI−

mutants generated during conjugative replication of a UV-damaged F lac plasmid [36]. A simple hypothesis to account for the defect in SOS mutagenesis caused by these Pol V mutations is that they may modify their interaction with RecA protein, resulting in an enhanced antirecombination activity associated with a modified activity in translesion synthesis. Alteration of Pol V DNA polymerase activity may have two distinct outcomes: either a global reduction in TLS resulting from a proportional decrease in both mutagenic and error-free bypass, or a change in the insertion and/or extension specificity of the polymerase leading to modification of the ratio between error-free and mutagenic TLS. To discriminate between these two possibilities, we used a Strand Segregation Analysis assay (SSA) to determine quantitatively (total TLS) and qualitatively (mutagenic versus error-free) TLS of a single (6-4)TT photoproduct [6,7,37]. Here, we report that TLS is markedly decreased in most of the Pol V mutants analyzed (8/9) with the exception of one UmuC mutant (F287L) that exhibits wild-type bypass activity. Somewhat unexpectedly, Pol V mutants that are partially deficient in TLS are more severely affected in mutagenic bypass compared to error-free synthesis. We discuss these results with respect to the location of the mutant Pol V amino acid changes in the 3D structure of UmuD [38] and the DinB/UmuC homologous protein Dpo4 of Sulfolobus solfataricus [39]. 2. Results and discussion 2.1. Replicative bypass of a single (6-4)TT lesion in cells expressing mutant Pol V polymerases To examine the ability of Pol V mutants to perform translesion synthesis of a single (6-4)TT UV photoproduct, we used the Strand Segregation Analysis assay (SSA) described in Fig. 1. The results of SSA performed on cells expressing a mutant Pol V polymerase are presented in Table 1. In these cells, the umuD C operon was expressed constitutively from a pSC101 plasmid at an elevated concentration (around 1000 UmuD C complexes per cell) [33,34] and it was not necessary to promote induction of SOS functions to observe TLS, the basal level of

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Fig. 1. Structure of the heteroduplex in the vicinity of the (6-4)TT photoproduct and quantification of mutagenic and error-free TLS in vivo by SSA. The top strand, containing the (6-4)TT photoproduct, is referred to as the target strand. The 3 -T of the (6-4)TT is part of a TAA ochre codon in phase with the lacZ reading frame. The bottom strand, containing a short sequence heterology across from the lesion, is referred to as the marker strand. Monomodified plasmids are derived from pCU plasmids [7]. When the heteroduplex construct replicates in host cells, any base substitution involving the 3 -T base of the (6-4)TT lesion will change the stop into an aminoacid that confers a [Lac+ ] phenotype to the corresponding colonies, while insertion of the ‘correct’ base (i.e. an ‘A’) opposite the 3 -T base of the lesion restores the TAA stop codon thus confering a [Lac− ] phenotype to the corresponding colonies. The frequency of mutagenic bypass of the (6-4)TT lesion is thus calculated as the ratio of blue colonies to the total number of AmpR colonies generated after electroporation of the host strain and plating on LB agar plates containing ampicillin (100 ␮g/ml) plus 5-bromo-4-chloro-3-indolyl ␤-d-galactoside (X-Gal, 40 ␮g/ml). The frequencies of error-free translesion synthesis and damage avoidance are measured using the Strand Segregation Analysis assay (SSA) by replicating a sample of white colonies onto nylon membranes [6]. After overnight incubation, the colonies are hybridized with radiolabelled 20-mer oligonucleotide target probe (5 -AGTCGCAAGTTAACACGGAC-3 ) and marker probe (5 -AGTCGCAACGCAGAACACGG-3 ) at the discriminating temperatures of the oligonucleotides (60 and 64 ◦ C, respectively) as previously described [6,7]. Filters are subsequently analyzed using a Molecular Dynamics Phosphorimager. Colonies hybridizing to both target and marker probes are identified as generated by error-free TLS events, while colonies hybridizing only to the marker probe are scored as damage avoidance events. It should be noticed that error-free and mutagenic TLS are likely to be two different outcomes of a single mechanism of replication of damaged DNA that carries a certain probability of error.

RecA being sufficient for this process (around 9000 RecA monomers per cell) [29,40]. We tested nine distinct Pol V mutants (four and five with a single point mutation in UmuD and UmuC, respectively) in comparison to wild-type Pol V. The extent of mutagenic TLS was determined as the fraction of blue colonies on X-gal containing indicator plates (i.e. Lac+ ) among the total number of colonies.

Except for strains expressing UmuCF287L , there was a general decrease in mutagenic translesion synthesis in all Pol V mutants, umuDG25D , umuDE35K and umuCF10L , umuCY270L , umuCK277E being the most severely affected (Table 1; Fig. 2). Fig. 2 shows a comparison of the ability of a mutant Pol V polymerase to promote mutagenic bypass of a (6-4)TT lesion with its ability to promote mutagenesis

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Fig. 2. The ability of a mutant Pol V polymerase to promote mutagenic bypass of a (6-4)TT lesion correlates with its ability to promote mutagenesis on a UV-damaged F lac plasmid. (A) Donor E. coli GY8630: recA13 uvrB501 StrS (F42-10: F::Tn10 lac+ ) [33] were exposed to 0 or 20 J/m2 of UV light and mated with derivatives of E. coli GY8352: F− lac-pro umuDC StrR [33] containing plasmid pGB2 (䊐) or pGY9864: o1 c umuD C (䊏) [40] or pGY9864 derivatives constitutively expressing the indicated UmuD or UmuC mutant proteins in conjunction with their respective wild-type partners ( ). The matings were performed on a filter at a 1.5:1 donor/recipient ratio. After 90 min, the mating mixtures were vigorously shaken in TMG buffer and diluted samples were plated on plates containing streptomycin, tetracycline and X-Gal to score the lacI− mutants among the sexductants (upper panel) (data from Sommer et al. [36]) (B) A heteroduplex construct containing a single (6-4)TT lesion in the lacZ gene was used to transform, via electroporation, E. coli MGZ strain: (umuDC)595::cat lacZM15 [17] derivatives carrying plasmid pGB2 (䊐) or pGY9864: o1 c umuD C (䊏) or pGY9864 derivatives encoding the indicated UmuD or UmuC mutant proteins in conjunction with their respective wild-type partners ( ). The frequency of base substitutions was calculated as described in Fig. 1.

on a UV-damaged F lac plasmid. Interestingly, we observed a positive correlation between the efficiency of mutagenic translesion synthesis in the two assays, despite the differences in the DNA substrates: (i) a double-stranded plasmid containing a single (6-4)TT lesion located in a particular sequence context in the SSA assay, or (ii) an F lac episome containing single-stranded discontinuities generated during conjugal replication and located opposite UV-lesions positioned in different sequence contexts [36]. This positive correlation between the results obtained in these assays validates the use of each of them to

test the ability of bypass polymerases to promote mutagenic translesion synthesis of a UV-lesion. To measure TLS accurately, between 1000 and 2000 independent [Lac− ] colonies for each Pol V expressing strain were tested, using hybridization with radiolabelled 20-mer oligonucleotides probes complementary to the target strand (target probe) and to the marker strand (marker probe). Except for strains expressing UmuCF287L , we observed a general decrease in error-free translesion synthesis in the Pol V mutants (Table 1) consistent with the general decrease found in mutagenic translesion synthesis (Table 1; Fig. 2). This

S. Sommer et al. / DNA Repair 2 (2003) 1361–1369 Table 1 Replicative bypass of a single (6-4)TT lesion in hosts expressing mutant Pol V polymerases Plasmid umuDC allele oC umuD C+ oC umuDG25D C+ oC umuDS28T C+ oC umuDP29L C+ oC umuDE35K C+ oC umuD CF10L oC umuD CY270L oC umuD CK277E oC umuD CF287L oC umuD CK342Q

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2.2. Localization of Pol V amino acid changes on the 3D structure of UmuD and the DinB/UmuC homologous protein Dpo4 of S. solfataricus

Translesion synthesis (%) Mutagenic 12.8 (277/2168) 0.9 (15/1769) 5.1 (82/1607) 3.7 (65/1727) 1.0 (17/1758) <0.05 (0/1944) 0.2 (3/1786) 0.25 (4/1744) 13.7 (126/917) 7.7 (142/1832)

Error-free 3.7 (81/2168) 1.0 (18/1769) 1.6 (26/1607) 1.6 (28/1727) 0.9 (16/1758) <0.05 (0/1944) 0.05 (1/1786) 0.5 (9/1744) 4.3 (39/917) 2.2 (40/1832)

Total 16.5 1.9 6.7 5.3 1.9 <0.05 0.25 0.75 18.0 9.9

A heteroduplex construct containing a single (6-4)TT lesion in the lacZ gene [7] was used to transform, via electroporation, E. coli MGZ (umuDC)595::cat, lacZM15 [17] derivatives containing plasmid pGB2 or pGY9864: oc1 umuD C or pGY9864 derivatives encoding the indicated UmuD or UmuC mutant proteins in conjunction with their respective wild-type partner (column 1) [36]. The frequencies of mutagenic (column 2) and error-free TLS (column 3) were determined as described in Fig. 1. The frequencies of error-free TLS were corrected by substracting error-free TLS found in a (umuDC) host (in fact, only 0.8% corresponding to 12 error-free TLS events among 1522 white colonies). This background of error-free TLS measured in a (umuDC) host could correspond to a contamination of the substrate by a small fraction of photoproduct free plasmids that are intrinsically non-mutagenic.

can be clearly seen in Fig. 3 in which the mutants are ranged according to their increasing deficiency in mutagenic (hatched bars) or accurate TLS (closed bars). Interestingly, in mutants umuDP29L , umuDE35K , umuDG25D , umuCK277E , the relative proportion of mutagenic and accurate TLS is altered. Indeed, they were more severely affected in mutagenic than in error-free TLS (Fig. 3). While the fraction of accurate TLS relative to the total TLS was 24% in cells expressing the wild-type Pol V, this value increased to 30% in umuDP29L and to around 50% in umuDE35K , umuDG25D , and umuCK277E . The qualitative and quantitative alterations in translesion synthesis displayed by the umuDG25D , umuDP29L , umuDE35K , and umuCK277E mutant polymerases suggest that these mutations might affect the specificity of nucleotide incorporation of the polymerase or its ability to extent a 3 OH terminus when a base other than A has been incorporated opposite to the (6-4)TT UV photoproduct.

The 3D crystal structure of the UmuD dimer was solved by Peat et al [41] and more recently the 3D structure of UmuD dimer in solution was determined by NMR [38]. All the UmuD amino acid changes like G25D, S28T, P29L and E35K that enhance UmuD2 C-dependent inhibition of homologous recombination are clustered in the N-terminal arm of UmuD . Since this N-terminal arm is disordered and solvent exposed [38,41–43], a structure/function analysis of this domain cannot be performed. Nevertheless, the fact that all the mutations are clustered in this region suggests that UmuD2 C could bind to the RecA filament using its free arms, and could competitively inhibit binding of dsDNA for homologous recombination as suggested by Ferentz et al. [38]. Mutations in this UmuD region could also modify the preferential binding of the UmuD C complex to the tip of a RecA filament required for TLS and the complex could be sequestered at inappropriate sites within the deep helical groove of a RecA polymer and kept away from the lesion because of its strengthened interaction with RecA. The 3D structure of the DinB/UmuC homologue Dpo4 from S. solfataricus P2 was recently solved [39]. Sequence-based alignments of UmuC with the 3D structure of Dpo4 [39,44] shows that Y270 (A268 in Dpo4), K277 (K275 in Dpo4, F287 (H285 on Dpo4), and K342 (K339 on Dpo4) are located on the surface of the protein (Fig. 4B and C) [45]. The four amino acids are close to each other and all lie in the putative little finger domain of UmuC. The “little finger” domain is a distinct feature of the Y-family of DNA polymerases [39] (see [13] for a recent minireview). The sequence corresponding to the little finger is not conserved among members of the Y-family bypass polymerases, though secondary structure predictions indicate that all of the members can potentially form the ␤␣␤␤␣␤ structure found in the little finger of Dpo4 [39]. This structure could be essential for efficient bypass of DNA lesions by the Y-family polymerases. Based on the ternary complex of Dpo4, F287 is located close to the template strand, while K342 interacts with the backbone of the primer (see Fig. 4C) [45]. However, only a minor effect on TLS is observed

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Fig. 3. Error free and mutagenic translesion synthesis are differentially affected in cells expressing different mutant Pol V polymerases. The efficiencies of mutagenic and error-free TLS, in hosts expressing the indicated Pol V mutants, were expressed as the percentage of the efficiencies of mutagenic and error-free TLS in bacteria expressing wild-type Pol V using results presented in Table 1.

in these mutants, suggesting that these substitutions are not critical for interaction with DNA. In contrast, the amino acid Y270L or K277E substitutions have a dramatic effect on TLS (Figs. 2 and 3). Amino acid Y270 is located in the hydrophobic core of the little finger domain and amino acid K277 lies within the DNA-binding cleft of the polymerase. The side chain of the Lys residue interacts with the backbone of the DNA template (Fig. 4B and C) [45]. Interestingly, another mutation (R279C) isolated by Woodgate et al. [46] in the same domain of UmuC, was also found to have a dramatic effect on SOS mutagenesis. Modification of these residues are likely to disrupt or alter interactions with DNA, reflecting the dramatic decrease in observed TLS in vivo (Table 1; Fig. 3). These amino acids could also have an indirect influence on bypass activity through the modified interaction of the mutant Pol V with the RecA filament and this could also account for the enhanced antirecombination activity of the mutant Pol V. The most deficient Pol V mutant in which TLS was virtually abolished is umuCF10L (Fig. 3). The F10 amino acid (F11 in Dpo4) is conserved in several DNA

polymerases of the Y-family, including E. coli UmuC and DinB, S. solfataricus Dpo4, S. cerevisiae Pol ␩ and human Pol ␩, Pol ␬ and Pol ␫. In the structure of Dpo4 determined from the co-crystals of ternary complexes (Polymerase–Primer/Template-dNTP) [39], the corresponding F11 amino acid is the first residue of an alpha helix close to the adjacent beta strands containing the catalytic triad D7, D105 and D106 located in the active site palm domain of Dpo4 (Fig. 4C). The complete defect in TLS of the umuCF10L mutant, and the absolute conservation of this F amino acid in all the bypass polymerases, suggest a crucial role for this amino acid in maintaining the hydrophobic catalytic site of UmuC. In summary, according to their quantitative and qualitative alterations in TLS, the Pol V mutants studied here can be divided into three classes (i) those (umuDP29L , umuCF287S , umuCK342Q that retain all or most of the global TLS activity of Pol V (ii) those (umuDP29L , umuDE35K , umuDG25D , umuCK277E ) that, in addition to a reduction in global TLS, differ quantitatively from the wild type in the relative proportion of accurate versus mutagenic TLS, (iii) those

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(umuCF10L , umuCY270L ) that are almost completely deficient in mutagenic as well as in accurate TLS. In vivo and in vitro experiments point to RecA as the target of the Pol V antirecombination activity: (i) the RecA [UmuR ] amino acid changes enable

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the RecA protein to escape recombination inhibition by Pol V in vivo [29], (ii) a RecA [UmuR ] protein RecAS117F has been shown to interact poorly with the UmuD protein in vitro [47], (iii) the wild-type Pol V has been shown to bind directly to a RecA filament in vitro and to inhibit the RecA-mediated strand exchange reaction [32,35,48]. Therefore, the enhanced antirecombination activity of the Pol V mutants characterized here is likely to result from a stronger and modified interaction with a RecA::ssDNA nucleoprotein filament. However, we cannot exclude that an enhanced antirecombination activity of some Pol V mutants could result from an altered interaction with SSB. SSB has been shown to directly contact MucB, a plasmid UmuC homologue, and to play an essential role in TLS and in homologous recombination, by favoring the formation of the RecA::ssDNA nucleoprotein filament [49,50]. It has been proposed that a RecA filament behaves as a directional chaperone to position the Pol V complex directly at a lesion in order to allow essential interactions of Pol V with other components such as the ␤ clamp and SSB involved in TLS [31,32,51,52]. We would like to stress that except F10, all the other umuC

Fig. 4. Mapping of the Pol V amino acid changes onto the structures of UmuD and the UmuC homologue Dpo4. (A) Mapping of the UmuD amino acid changes in a UmuD dimer. The solution structure of the UmuD homodimer used in this figure was determined by Ferentz et al. [38] and deposited in the PDB database under the accession number 1I4 V. Mutant positions (G25, S28, P29 and E35) are highlighted in cyan. (B) Mapping of the UmuC amino acid changes on the structure of a S. solfataricus DinB/UmuC homologue Dpo4. The crystal structure of Dpo4, in a ternary complex with a DNA substrate and an incoming nucleotide, was solved by Ling et al. [39] and deposited in the PDB database under the accession number 1JXL. Dpo4 is shown in a ribbon representation with the backbone in white and the side chains of A268, K275, H285 and K339 (corresponding to Y270, K277, F287, and K342 in the E. coli UmuC protein) in cyan, F11 (corresponding to F10 in the E. coli UmuC protein) in yellow, and the catalytic triad D7, D105 and E106 (corresponding to D6, D101 and D102 in the E. coli UmuC protein) in red. The template strand is colored in pink, while the primer strand is represented in purple. (C) Close up of the little finger domain of Dpo4. The residues are numbered based on their respective position in the E. coli UmuC protein. All the amino acid changes (Y270, K277, F287 and K342) are located in the little finger domain of the polymerase, except for F10, which is located in the vicinity of the catalytic core. These figures were generated by PyMol Molecular Graphic software [54].

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mutations (Y270, K277, F287, and K342) are located within the little finger domain, near the consensus peptide (AQLNLF) that is directly involved in targeting Pol V to the ␤-clamp [52,53]. It may therefore be possible that a modified interaction between Pol V and RecA might result in an altered interaction of Pol V with the ␤-clamp and consequently affect translesion synthesis. Resolution of the underlying mechanisms of TLS deficiency awaits further biochemical and electron microscopic characterization of these mutants.

Acknowledgements We are grateful to Raymond Devoret for his constant interest in this work. We thank Michael DuBow for critical reading of this manuscript. Grants from Association pour la Recherche contre le Cancer (No 4360), the Ministère de l’Education Nationale, de la Recherche et de la Technologie (AS Radiobiologie 98-15) and from HFSP (RG 0351/1998-M) provided invaluable help. We thank the Institut Curie and the Centre National de la Recherche Scientifique for providing funds. This work was carried out in compliance with the current laws governing genetic experiments in France.

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