The Relationship Between Dntp Pool Levels and Mutagenesis in an Escherichia Coli NDP Kinase Mutant

The relationship between dNTP pool levels and mutagenesis in an Escherichia coli NDP kinase mutant

Jared Nordman and Andrew Wright*

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111

Edited by Jonathan Beckwith, Harvard Medical School, Boston, MA, and approved May 6, 2008 (received for review March 27, 2008) Loss of nucleoside diphosphate kinase (Ndk) function in Escherichia The human genome contains eight Ndk paralogues, Nm23 coli results in an increased frequency of spontaneous mutation and H1–H8, which have been implicated in multiple cellular pro- an imbalance in dNTP pool levels. It is presumed that the imbalance cesses. For example, human NDP kinase (nm23-H1) was initially in dNTP pool levels is responsible for the mutator phenotype of an identified as a suppressor of tumor metastasis by using meta- E. coli ndk mutant. A human homologue of Ndk and potential static melanoma cell lines (17). Nm23-H2 has been shown to suppressor of tumor metastasis, nm23-H2, can complement the cleave DNA in a mechanism similar to that of the AP lyase family mutagenic phenotype of an E. coli ndk mutant. Here, we show that of DNA repair enzymes, and directly regulate expression of the the antimutagenic property of nm23-H2 in E. coli is independent of c-MYC oncogene (18, 19). Mutational loss of NDP kinase dNTP pool levels, indicating that dNTP pool imbalance is not function in Drosophila leads to developmental defects (20). responsible for the mutator phenotype associated with the loss of Interestingly, restoration of NDP kinase enzymatic activity is ndk function. We have identiﬁed multiple genetic interactions necessary but not sufficient to complement this mutant pheno- between ndk and genes involved in the metabolism of dUTP, a type (21). potentially mutagenic precursor of thymidine biosynthesis. We E. coli ndk mutants have an alteration in dNTP pool levels, show that loss of ndk function is synergistic with a dut-1 mutation which is thought to give rise to a weak mutator phenotype and synthetically lethal with the loss of thymidine kinase function. associated with the loss of ndk function (13). Expression of Our results suggest that Ndk prevents the accumulation of dUTP in human nm23-H2 complements the mutagenic phenotype of an vivo. Based on these results and biochemical studies of Ndk, we E. coli ndk mutant (22). Ndk has also been shown to cleave DNA MICROBIOLOGY propose that the mutagenic phenotype of an ndk mutant is caused and functionally interact with the uracil glycosylase, Ung (23, by excess misincorporation of uracil in place of thymidine com- 24). The interaction between Ndk and Ung is reported to lower bined with a defect in the uracil base excision pathway. the Km of Ung for its substrate, uracil containing DNA (23). Ung is responsible for the excision of uracil, which can be misincor- DNA replication ͉ dNTP synthesis porated in place of thymidine (25). Although dUTP is an essential precursor in the de novo synthesis of thymidine, it is n Escherichia coli the DnaA protein initiates bidirectional necessary that its concentration be kept low to prevent such IDNA replication at a unique origin of replication, oriC,ina misincorporation (26). Indeed, because of the action of the cell-cycle dependent manner (1). In addition to its role as the essential enzyme dUTPase, which converts dUTP to dUMP, the initiator of DNA replication, DnaA is a transcriptional regulator, dUTP concentration is estimated to be Ͻ1% of the total dTTP coordinately regulating the expression of numerous genes with concentration (27, 28). respect to the cell cycle (2, 3). One set of genes directly regulated In an effort to better understand the role of Ndk in DNA by the DnaA protein encodes the enzyme ribonucleotide reduc- replication and mutagenesis, we used a human nm23-H2 con- tase, known as NrdAB or RNR (4). Because reduction of struct to determine whether its antimutagenic effect was caused nucleoside diphosphates (NDPs) is the rate-limiting step in the solely by NDP kinase phosphotransferase activity. Here, we production of dNTPs, regulated expression of RNR with respect show that human nm23-H2 is able to complement the mutagenic to initiation of DNA replication ensures sufficient dNTPs will be phenotype of an E. coli ndk mutant without restoring dNTP pool produced during the replication period (5). levels. This finding provides definitive evidence that the dNTP dNTP pools are limiting for DNA replication, their levels in pool imbalance associated with loss of ndk function is not the cell being only Ϸ1% of the amount necessary for a single responsible for its mutator phenotype. Furthermore, we show round of chromosomal replication (1, 6). Furthermore, reducing that ndk genetically interacts with dut and tdk, two genes the concentration of a single dNTP can cause a decrease in the specifically involved in dUTP/dUMP metabolism, and provide rate of replication fork progression in vivo (7). Improperly evidence that increased mutagenesis in an ndk mutant is likely regulated synthesis of dNTPs by altered expression and/or occurring from misincorporaton of uracil in place of thymidine. activity of RNR in Saccharomyces cerevisiae can be mutagenic Based on these results, we propose that Ndk functions to prevent and affect cell-cycle progression (8, 9). excess dUTP accumulation in vivo and that E. coli Ndk and We have previously shown that mutations in ndk, which human Nm23-H2 possess DNA repair activities specific to uracil encodes nucleoside diphosphate kinase (Ndk), decrease the base excision repair. relative rate of replication fork progression in vivo, suggesting an important link between proper regulation of dNTP synthesis and replication fork elongation (10). NDP kinases are a broad and Author contributions: J.N. and A.W. designed research, performed research, contributed well conserved family of enzymes present in all domains of life. new reagents/analytic tools, analyzed data, and wrote the paper. In vitro, NDP kinases are capable of generating all (d)NTPs from The authors declare no conﬂict of interest. their corresponding (d)NDPs through a well characterized phos- This article is a PNAS Direct Submission. photransferase reaction (11). Based on the ability of NDP kinase Freely available online through the PNAS open access option. to generate all (d)NTPs in vitro it is assumed to be the major *To whom correspondence should be addressed. E-mail: [email protected]. enzyme involved in dNTP synthesis in vivo. But NDP kinases This article contains supporting information online at www.pnas.org/cgi/content/full/ from many organisms are dispensable; their loss has little or no 0802816105/DCSupplemental. effect on cell growth (12–16). © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802816105 PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 10197–10202 Downloaded by guest on September 24, 2021 initiation of DNA replication. Furthermore, we showed that loss of Ndk function results in a decreased rate of replication fork progression, which is likely responsible for the suppression of overinitiation (10). To test whether mutagenesis has a role in this novel phenotype associated with loss of Ndk function in E. coli, we introduced the pnm23-H2 plasmid into a dnaA(cos) ndk double mutant to ask whether nm23-H2 could complement the double mutant and restore cold sensitivity. As shown in Fig. 1B, the pnm23-H2 construct did not restore cold sensitivity to the dnaA(cos) ndk double mutant. This finding suggests that Nm23-H2 lacks an activity associated with E. coli Ndk function. Loss of Ndk in E. coli results in an imbalance in dNTP pool levels, which is thought to give rise to the mutator phenotype. Specifically, the level of dCTP was reported to increase Ϸ20-fold and the level of dGTP Ϸ7-fold with only modest changes in dATP and dTTP when compared with WT cells (13). However, the fluctuation in dNTP pool levels and their role in mutagenesis became unclear when it was reported that strain background differences affected dNTP pool levels, but not the frequency of Fig. 1. Human nm23-H2 does not fully complement an E. coli ndk mutant. spontaneous mutagenesis in an ndk mutant (29). Furthermore, (A) Frequency of spontaneous Rif resistance in four to seven independent mutational bias in an ndk mutant is inconsistent with the notion cultures for each strain tested. Median and interquartile ranges are presented. that changes in dNTP pool levels, specifically an increase in The median values are 2.5, 38.46, 1.38, and 2.10 for WT, ndk, ndk/pndk, and dCTP and dGTP, are responsible for mutagenesis (22). ndk/pnm23-H2, respectively. (B) Cells of a dnaA(cos) or dnaA(cos) ndk double mutant harboring the indicated plasmid were grown to log phase at 42°C, 10- To address these issues, dNTP pool levels were assayed in WT fold serially diluted, and incubated at 42°C or 30°C for 18 or 24 h, respectively. and ndk mutant cells complemented with either E. coli ndk or nm23-H2. As shown in Table 1, loss of ndk function gives rise to a modest change in dNTP pool levels, with Ϸ2- to 3-fold Results increases in dCTP, dTTP, and dGTP and a Ϸ4-fold decrease in dNTP Pool Imbalances Are Not Responsible for Increased Mutation dATP. These values are consistent with results reported by Shen Frequency in an ndk Mutant. To gain further insight into the role et al. (29) and support the notion that dNTP pool measurements of Ndk in DNA replication and mutagenesis, we fused the human are affected by strain background differences. Imbalances in ndk homologue, nm23-H2,totheE. coli ndk promoter and dNTP pool levels could be corrected by providing E. coli ndk in trans, but not nm23-H2. These results demonstrate that an cloned this construct in a low-copy plasmid (Ϸ10 copies per cell). imbalance in dNTP pool levels is not responsible for the Nm23-H2 has been shown to complement the mutator pheno- increased frequency of spontaneous mutation in an ndk mutant. type of an E. coli ndk mutant (22). To test the functionality of dNTPs are limiting for DNA replication, the dNTP pool being our nm23-H2 construct, we introduced the pnm23-H2 plasmid only Ϸ1% of what is needed for one round of chromosomal into an E. coli ndk mutant and measured the frequency of replication (1, 6). Lack of complementation of the dnaA(cos) spontaneous mutagenesis (see Materials and Methods). The ndk double mutant by nm23-H2 (Fig. 1B) suggests that the frequency of spontaneous rifampicin (Rif) resistance in the ndk reduction in the dATP pool level is responsible for the decreased Ϸ mutant was 6- to 15-fold increased relative to that in WT cells rate of replication fork progression associated with the loss of (Fig. 1A and Table 1). Expression of E. coli ndk and human ndk function. Limitation of the intracellular thymidine pool is nm23-H2 in trans reduced the frequency of spontaneous mu- known to have a similar effect on the rate of replication fork tagenesis to levels comparable with that of WT cells (0.55- and progression (7). 0.84-fold increase, respectively; Fig. 1A and Table 1). These Because an ndk mutant strain expressing nm23-H2 behaves results confirm that nm23-H2 complements the mutator pheno- like an ndk null mutant with respect to dNTP pool levels, but no type of an ndk mutant, as previously reported, and indicate that longer has the mutator phenotype of an ndk mutant, we asked our nm23-H2 construct is functional (Fig. 1A). whether we could detect NDP kinase activity in cellular extracts We previously identified loss of Ndk function as a suppressor derived from the nm23-H2-expressing strain (see Materials and of dnaA(cos)-mediated overinitiation that acts independently of Methods). Nm23-H2-expressing cells had only Ϸ2-fold increased

Table 1. dNTP pool imbalances are not responsible for the increased mutagenic frequency in an ndk mutant Assay WT ndk ndk/pndk ndk/pnm23-H2

dCTP* 1 2.38 (Ϯ0.58) 1.02 (Ϯ0.26) 2.71 (Ϯ0.45) dGTP* 1 2.19 (Ϯ0.32) 1.49 (Ϯ0.17) 3.06 (Ϯ0.49) dATP* 1 0.27 (Ϯ0.02) 1.08 (Ϯ0.19) 0.76 (Ϯ0.16) dTTP* 1 2.76 (Ϯ0.90) 1.47 (Ϯ0.20) 2.69 (Ϯ0.26) RifR /108 CFU† 1 15.38 0.55 0.84 NDK activity‡ 1 0.091 (Ϯ0.02) 0.797 (Ϯ0.13) 0.167 (Ϯ0.03)

*For relative dNTP pool measurements, values represent the mean of three independent cellular extracts. The values in parentheses indicate the SEM. †For RifR resistance frequencies, values represent the fold differences relative to WT of four to seven independent cultures. The actual median values are represented in Fig. 1. ‡For NDP kinase activity, values represent the mean of three independent extract preparations. Values in parentheses indicate the SEM.

10198 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802816105 Nordman and Wright Downloaded by guest on September 24, 2021 Table 2. Frequency of spontaneous Rif resistance Table 3. Relative dNTP pool measurements Strain RifR CFU/108 CFU* Fold increase† dut-1 dNTP WT ndk dut-1 dut-1 ndk ndk ung WT 1.67 (0.48–3) 1 ndk 10.63 (7.6–29.3) 6.37 dCTP 1 4.51 (Ϯ0.46) 1.27 (Ϯ0.06) 7.05 (Ϯ0.80) 8.32 (Ϯ1.04) dut-1 1.28 (0.39–3.2) 0.77 dGTP 1 1.86 (Ϯ0.13) 0.69 (Ϯ0.10) 1.03 (Ϯ0.06) 1.36 (Ϯ0.17) dut-1 ndk 5,005 (3,682Ϫ15,623) 2,997‡ dATP 1 0.79 (Ϯ0.05) 1.64 (Ϯ0.11) 0.54 (Ϯ0.08) 0.79 (Ϯ0.14) dut-1 ung 3.39 (0.8–10.2) 2.03 dTTP 1 3.68 (Ϯ0.16) 1.78 (Ϯ0.30) 3.87 (Ϯ0.05) 4.34 (Ϯ0.83) dut-1 ndk ung 524 (428–3,348) 314‡ Values represent the average of three independent cellular extracts. The ung 2.48 (1.99–12.8) 1.49 values in parentheses indicate the SEM. ung ndk 8.90 (6.8–11.3) 5.33 dut-1 ndk/pndk 1.04 (0.66–1.52) 1 dut-1 ndk/pnm23-H2 7.69 (5.96–9.56) 7.69 To determine whether the synergistic increase in mutation dut-1 ndk/pBAD33 3,703 (3,272–12,151) 3,578 frequency in the dut-1 ndk double mutant was caused by *Values represent the median from six to 12 independent cultures for each excessive uracil incorporation, we introduced an ndk mutation strain tested and parentheses represent the 95% CI of the median or the 85% into a dut-1 ung double mutant. The resulting triple mutant had CI of the median for the complemented strains. a substantial increase in mutation frequency (Ϸ314-fold relative †Fold increase relative to WT (ED8566). to WT; Table 2) but had a Ϸ10-fold reduced frequency com- ‡A t test indicates P ϭ 0.0065 when comparing the dut-1 ndk double mutant pared with the dut-1 ndk double mutant (P ϭ 0.0065). Taken and the dut-1 ndk ung triple mutant. together, these results suggest that the synergistic effect on mutagenesis observed in the dut-1 ndk double mutant is likely NDP kinase activity compared with the ndk mutant, whereas caused by replication intermediates generated from the uracil the ndk strain complemented with E. coli ndk showed NDP excision process, because stable incorporation of uracil into kinase activity similar to that of WT cells (79% relative to WT; DNA can partially alleviate this mutagenic effect. Table 1). Taken together, these results indicate that the anti- To further address the relationship between dNTP pool mutagenic activity of nm23-H2 is independent of dNTP pool imbalance and mutagenesis, we measured the dNTP pool levels in ndk, dut-1, dut-1 ndk, and dut-1 ung ndk mutant cells and

levels and NDP kinase activity. Thus, low-level expression of MICROBIOLOGY nm23-H2 does not provide all of the functions missing in an ndk compared them with those of WT cells. As previously shown, ndk mutant. cells had only a slight alteration in dNTP pool levels relative to WT cells (Tables 1 and 3). Also, a dut-1 mutation had only a A dut-1 Mutation Synergistically Increases the Spontaneous Mutation modest effect on dNTP pool levels, resulting in a slight increase Frequency of an ndk Mutant. Biochemical studies of E. coli Ndk in dCTP, dATP and dTTP (1.27-, 1.64-, and 1.78-fold, respec- have identified a functional interaction between Ndk and uracil tively) and a slight decrease in dGTP (31% reduction). The dut-1 glycosylase, Ung (23). This interaction has been shown to lower ndk double mutant and the dut-1 ndk ung triple mutant had very similar dNTP pool levels, a Ϸ2-fold increase in dCTP being the the Km of Ung for its substrate, uracil-containing DNA, which led to the hypothesis that Ndk could have a role in repair of only significant difference when compared with ndk mutant cells uracil-containing DNA. Human Nm23-H2 has been shown to (Table 3). Although there was no difference in the dNTP pool cleave DNA by a mechanism similar to that of AP lyases, and levels between the double and triple mutant strains, there was a E. coli Ndk has also been shown to cleave DNA, but the specific Ϸ10-fold difference in their mutation frequency. amino acid residue necessary for this activity has not yet been To further analyze the severe mutagenic effect in the dut-1 ndk defined (18, 24). However, the residue necessary for Nm23-H2 double mutant, we asked whether there was a mutational bias DNA cleavage activity (K11) is conserved in E. coli Ndk (K12), associated with that strain. Mutational bias was previously suggesting both enzymes cleave DNA in a similar manner. Taken determined for an ndk mutant by using mutation to Rif resis- together, these results suggested to us that Ndk could have a role tance (22), but the limited range of mutations known to give rise in uracil metabolism. To test this hypothesis, we combined an to Rif resistance led us to screen instead for lac constitutive ndk mutation with known mutations that affect uracil metabo- mutants that result from loss of lacI function (33). If the lism and assayed the level of spontaneous mutagenesis in the mutational event that gives rise to the lacconst phenotype is caused resulting strains. by excessive dUTP incorporation and uracil excision, mutations For the de novo synthesis of thymidine, dCTP is deaminated affecting thymidine residues (AT base pairs) should be overrep- to form dUTP, which is rapidly hydrolyzed to dUMP by the resented. Indeed, of 47 independent lacIconst mutants isolated, enzyme dUTPase (Dut). dUMP is then methylated to form 92% (43/47) resulted from mutations originating at an AT base dTMP (26). Dut is an essential protein (30, 31), but a dut-1 pair with only 2% (1/47) originating at a GC base pair (Table 4). mutant strain, which retains only 2–3% the activity of WT Dut activity is capable of supporting growth (30, 32). A dut-1 mutant Table 4. Bias of mutations in the lacI gene leading to a lacconst has a modest effect on spontaneous mutation frequency and loss ؊ of Ndk function results in Ϸ6-fold increase in mutation fre- phenotype in a dut-1 ndk double mutant quency compared with WT cells (Table 2). An ndk dut-1 double Base change Occurrence, % AT or GC bias, % mutant, in contrast, was found to have a Ϸ3,000-fold increase in 3 mutagenesis (Table 2). This synergistic genetic interaction sug- AT GC 79 (37/47) 3 gests that loss of Ndk function results in increased levels of dUTP AT CG 4 (2/47) 92 AT3TA 9 (4/47) and that normal Dut activity is necessary to maintain the dUTP 3 pool at a lower level. Expression of Ndk in trans completely GC AT 2 (1/47) GC3TA 0 (0/47) 2 reversed this increase in mutation frequency, whereas reversal by 3 Nm23-H2, expressed in trans, was incomplete (Ϸ8-fold increase GC CG 0 (0/47) in mutagenesis relative to WT; Table 2). These results are Other* 6 (3/47) 6 consistent with the idea that Nm23-H2 functions independently The lacI gene ϭ 43.7% AT, 56.3% GC. n ϭ 47. of dNTP pool regulation. *Other indicates (Ϫ1) frameshifts or a nucleotide insertion.

Nordman and Wright PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 10199 Downloaded by guest on September 24, 2021 above, we introduced a dcd (dCTP deaminase) null mutation into an ndk mutant. Approximately 75% of the dUTP in the cell is derived from deamination of dCTP to form dUTP, a process catalyzed by dCTP deaminase (26). To our surprise, we observed a genetic interaction between dcd and ndk. Cells of a dcd ndk double mutant gave rise to small heterogeneous colonies, whereas neither single mutant did so (Fig. S2). Furthermore, propagation of a dcd ndk double mutant rapidly gave rise to nonmutagenic, fast-growing derivatives, likely because of extra- genic suppression (unpublished data). Discussion Our motivation for studying the role of NDP kinase in DNA replication and mutagenesis in E. coli has been 2-fold. First, it provides a genetically tractable system to study how dNTP synthesis and dNTP pool imbalances affect DNA replication and mutagenesis. Second, it serves as a model system to study the Fig. 2. Loss of tdk function is synthetically lethal with loss of ndk function, physiological role of Ndk in vivo, which is apparently more which is only partially complemented by human nm23-H2. A single represen- complex than predicted, based solely on the known biochemical tative P1 transduction quantifying the efﬁciency that an ndk::EZ-Tn5 allele properties of Ndk. was transduced into a ⌬tdk mutant harboring the indicated plasmids is shown. Studies of E. coli ndk mutants led to the paradigm that dNTP A xerD::EZ-Tn5 allele was used as a control for P1 transduction frequencies. pool imbalances are responsible for the increased frequency of * indicates no colonies were detected. spontaneous mutagenesis (13). Our results indicate that expression of the human Ndk homologue, Nm23-H2, can complement the mutagenic phenotype of an E. coli ndk mutant, but is unable Loss of Thymidine Kinase Function Is Synthetically Lethal with the Loss to correct the imbalance in dNTP pool levels. Therefore, dNTP of Ndk Function. Results presented in this study, as well as pool imbalance is not responsible for the increased mutagenesis biochemical studies by Goswami et al. (23), indicate that Ndk has seen in an ndk mutant. a direct role in uracil metabolism. Based on this hypothesis we In attempts to address the role of Ndk in mutagenesis, we have performed a genetic screen to identify genes that are syntheti- uncovered multiple genetic interactions between ndk and genes cally lethal with loss of ndk function to better understand the involved in the metabolism of dUTP that suggest that Ndk physiological role of Ndk in vivo. For this screen we used a miniF prevents the formation of excess dUTP in vivo. We propose that plasmid that is unstable in the absence of antibiotic selection; an intermediate generated by the excision of uracil is responsible plasmid pJN65 contains the lacZ gene and the ndk gene under for mutagenesis in an ndk mutant, which is exacerbated when the control of its own promoter. JN319/pJN65 cells were subjected Ͻ Ͼ dUTP pool is elevated by a dut-1 mutation. AP (abasic) sites are to EZ::TN KAN-2 insertional mutagenesis (Epicentre) known to be highly mutagenic intermediates of the uracil and then plated on LB medium supplemented with kanamycin excision pathway and could provide a mechanistic explanation Ϸ and X-Gal. A total of 60,000 colonies from two independent for the increase in mutagenesis. In support of this model, stable experiments were screened for nonsectored solid blue colonies. uracil incorporation in a dut-1 ndk ung triple mutant displays a Four mutations were identified in this primary screen, and two reduction in mutagenesis when compared with the dut-1 ndk were verified by their inability to backcross the EZ-Tn5 insertion double mutant. The fact that the dut-1 ndk ung triple mutant still into the parental ndk strain (JN319). Sequence analysis showed has an elevated mutation frequency likely reflects an increase in that the mutations were caused by independent insertion events dUTP pool levels caused by the combination of a dut-1 and ndk within the tdk gene, which encodes thymidine kinase. mutation. These observations, in combination with the known To validate the synthetic lethal interaction between ndk and biochemical interaction of Ndk with Ung (23), and the fact that tdk, we constructed a strain containing an unmarked deletion of Ndk is able to cleave DNA (24), suggest that Ndk is at the the tdk ORF (see Materials and Methods) and transformed this interface of dUTP formation and uracil base excision repair (Fig. strain with plasmids expressing either ndk or nm23-H2 or with 3). Attempts to measure the frequency of uracil incorporation the plasmid vector alone. We then attempted to introduce an ndk into DNA in the dut-1 ndk double mutant strain by using a null allele, ndk::EZ-Tn5, into these strains by P1 transduction. A sensitive plasmid based assay developed by Kouzminova and xerD::EZ-Tn5 allele was used as a control for P1 transduction Kuzminov (34) were unsuccessful because of the high level of efficiency. Consistent with a synthetic lethal effect, efficient plasmid instability in this strain background (unpublished data). transduction yielding the tdk ndk double null mutant occurred If mutagenesis were occurring through uracil misincorpora- only in the case where Ndk was expressed in trans from the tion in place of thymidine and/or defects in excision of uracil, we plasmid (Fig. 2). In the case of cells expressing Nm23-H2, a would expect an overrepresentation of mutations originating at reduced number of transductants, forming smaller than normal AT base pairs, because U:G mismatches and abasic sites are both colonies was obtained, suggesting that Nm23-H2 only partially mutagenic. Indeed, 92% of all of the mutations we identified complemented the ndk/tdk synthetic lethal phenotype (Fig. 2). were caused by mutational events at AT base pairs. These data Furthermore, the Nm23-H2-complemented tdk ndk strain dis- agree with an analysis of mutational bias in an ndk mutant by played a temperature-sensitive growth defect when plated at Miller et al. (22), who also found that ndk mutants are hyper- 42°C [supporting information (SI) Fig. S1]. mutable upon loss of mismatch repair (MMR) activity. Excess Thymidine kinase is able to use dU or dT interchangeably. uracil misincorporation could give rise to U:G mispairs, which However, the tdk/ndk synthetic lethality likely reflects a dUMP- would subsequently be recognized by the MMR system. ndk specific interaction, because loss of thymidylate synthase (thyA) mutants and ndk mutS double mutants have nearly identical or thymidine phosphorylase (deoA) function had no effect when mutational biases, which supports this hypothesis. But, we cannot combined with an ndk mutation (data not shown). rule out the possibility that the dNTP pool imbalance in an ndk In an attempt to determine whether suppression of dUTP mutant gives rise to a low level of mispairs that are efficiently formation affected any of the genetic interactions described detected by the MMR system. It is important to note that this low

10200 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802816105 Nordman and Wright Downloaded by guest on September 24, 2021 Given this fact, combined with the observation that loss of Ndk function has no effect on ribonucleotide pools, which are in vast excess over dNTP pools (13), we propose that NDP kinase is not the primary determinant of (d)NTP synthesis in vivo. Adenylate kinase of E. coli is known to be capable of synthesizing all (d)NTPs in vitro (36) and is thus a candidate for performing this function in vivo. Materials and Methods Bacterial Strains, Media. E. coli K-12 strains used in this study are listed in Table S2. All strains were grown in L broth (LB) or M9 minimal medium supplemented with tryptophan and glucose. P1 transductions were carried out as described (37). Kanamycin (Kan; 30 ␮g/ml), 10 ␮g/ml chloramphenicol, 10 ␮g/ml tetracycline, 10 mM uracil, 40 ␮g/ml X-Gal, 25–50 ␮g/ml ampicillin, 250 ␮M isopropyl-␤-D- Fig. 3. E. coli NDP kinase genetic and biochemical interactions suggest that thiogalactoside, and 100 ␮g/ml Rif were added when necessary. ndk could function at the interface between dNTP synthesis and base excision repair. The uracil glycosylase base excision repair pathway (Left) and the Plasmid Constructions. All restriction enzymes and T4 DNA Ligase were from pathway of de novo thymidylate biosynthesis pathway (Right) are shown. New England Biolabs. All primers for PCR were synthesized by Integrated DNA Arrows with asterisk indicate genetic interactions identified in this study. The Technologies (IDT), and DNA polymerase was Triple Master (Eppendorf). All Ndk/Ung functional biochemical interaction was identified by Goswami et al. DNA constructs were verified by DNA sequencing at the Tufts University Core (23). Additionally, Ndk and human Nm23-H2 have been shown to cleave DNA Facility. The nm23-H2 ORF was amplified from plasmid pQE60nm23-H2 (22) and suggested to do so in a mechanism similar to AP lyases (18). The schematic with the primers 1331 (5Ј-TTTACAGAGGTAAAAATGGCCAACCTG- representation of the uracil BER pathway was adapted from ref. 25. GAGCGCACC-3Ј) and 1333 (5Ј-TTATTCATAGACCCAGTCATGAGCAC-3Ј). The E. coli ndk promoter region was amplified by using the primers 1299 (5Ј- CTGGATCCCGCGACAGTGAAATTTGTCATGC-3Ј) and 1332 (5Ј-GGTTGGC- level of mispairing is distinct from the phenotypic mutagenesis CATTTTTACCTCTGTAAATTGTTCTGTTGTTG-3Ј). These two PCR products were seen in a MMR-proficient ndk mutant. We have found that used together in an overlapping primerless PCR and then supplemented with Nm23-H2 only modestly alleviates mutagenesis in an ndk mutS primers 1299 and 1333. The resulting PCR product containing the E. coli ndk double mutant (Table S1). Regardless, because all methods used promoter region fused to the nm23-H2 ORF was ligated into pCR2.1 TOPO TA vector (Invitrogen), resulting in plasmid pJN15. The ndkPr-nm23-H2 containing MICROBIOLOGY to detect mutational bias show an increase in mutations origi- HindIII and XbaI fragment from pJN15 was ligated into HindIII/XbaI-digested nating at AT base pairs it is likely that misincorporation of uracil pBAD33 (38), resulting in pJN22. To create an inducible tdk allele, tdk was is the underlying cause. cloned into plasmid pDSW204 (39). The tdk ORF was PCR-amplified by using Our finding that a mutation in the thymidine kinase gene, tdk,is primers 1520 (5Ј- CCTCTAGACATTGAGGGCCTGTGGCTGATG-3Ј) and 1521 (5Ј- synthetically lethal with an ndk mutation, supports our hypothesis GGAAGCTTTTAATCGTGGCGATGCCTTTCC-3Ј), digested with XbaI/HindIII, and that Ndk is involved in uracil metabolism. Under conditions where ligated into XbaI/HindIII-digested pDSW204. ϩ ϩ dUTP metabolism is compromised, Tdk is thought to degrade The unstable lac /ndk miniF plasmid (pJN65) was created by PCR ampli- Ј dUMP to dU to help decrease the dUTP pool (26). In support of fying the ndk gene and upstream promoter region with the oligo 1299 (5 - CTGGATCCCGCGACAGTGAAATTTGTCATGC-3Ј) and oligo 1515. The resulting this hypothesis, we find that tdk mutants are sensitive to sublethal PCR product was digested with BamHI and HindIII and ligated into BamHI/ doses of fluorodeoxyuridine (Fig. S3). Also, loss of tdk function is HindIII-digested pFZY1 (40). synthetically lethal with loss of recA function, and although the mechanism is not entirely clear, it is likely caused by alterations in Spontaneous Mutation Frequency Determination. To determine the spontane- dUTP/dUMP metabolism because amplification of tdk can sup- ous mutation frequency, independent colonies were used to inoculate cul- press dut-1 recA synthetic lethality (35). tures that were grown overnight at 37°C with aeration. Cultures were serially Why do ndk mutants have an imbalance in dNTP pool levels diluted in sterile saline and plated onto LB plates for total CFUs or LB plates but no effect on rNTP pools? To date we are unaware of a supplemented with 100 ␮g/ml Rif. The total number of RifR CFUs was divided proposed mechanism to explain the dNTP pool imbalance. by the total CFUs to determine mutation frequency. The mutation frequencies Based on the observation that Ndk likely acts to prevent dUTP for the dut-1 ndk double mutant (JN264) and the dut-1 ung ndk triple mutant (JN266) was determined by using independent purified transductants from a formation in vivo, either directly or indirectly, we can propose a cross of JN57 ϫ JN263 or JN57 ϫ JN265 and selecting for KanR (ndk::EZ-Tn5) mechanism leading to dNTP pool imbalances in an ndk mutant. transductants. Because of rapid suppression of the mutator phenotype it was Because the enzyme dCTP deaminase, which converts dCTP to necessary to limit manipulation of these strains. Complementation analysis of dUTP, is feedback-inhibited by dUTP, an increase in the dUTP the dut-1 ndk double mutant was carried out by crossing JN57 with JN263/ concentration would inhibit dCTP deaminase activity, leading pBAD33, JN263/pJN15, or JN263/pJN22, selecting for KanR, and assaying in- to an increase in dCTP pool levels. Alternatively, if an ndk dependent transductants. mutant accumulated excess dCTP, it would be deaminated, resulting in an increase in both dCTP and dUTP pool levels. dNTP Pool Measurements. Overnight cultures were diluted 1:200 in LB and Ϸ Both mechanisms would predict a concomitant increase in dTTP grown to an OD600 0.2. To ensure balanced growth, these cultures were then diluted 1:10 in 10 ml of LB and grown at 37°C to an OD600 Ϸ0.1. Cultures were formation, which is observed in an ndk mutant. dTTP is an then either immediately filtered through a 0.45-␮m nitrocellulose filter (Mil- allosteric regulator of RNR activity and would, in turn, alter the lipore) to collect the cells or chilled rapidly in an ice-water bath before specificity of RNR to reduce GDP to dGDP. Because dTTP is filtration. The total CFUs were determined for each culture, and RifR CFUs were continually being synthesized because of increased dUTP for- determined for highly mutagenic cultures. Three independent cultures were mation, insufficient dGTP would be available to specify the used for each strain. reduction of ADP, leading to a decrease in dATP pool levels. Immediately after filtration, filters were vortexed in 5 ml of ice-cold 60% This model is consistent with the dNTP pool measurements methanol and placed at Ϫ20°C overnight. The methanolic suspension of cells associated with loss of Ndk function presented in this study and was boiled for 5 min and cells were removed by centrifugation. Because of those reported by Mathews and coworkers (29). volume differences in the final evaporation step, extracts were assayed for their absorbance at OD260 to quantitatively compare the nucleotide concen- NDP kinases are able to synthesize all (d)NTPs in vitro, but trations between each extract. loss of Ndk function in organisms as diverse as E. coli, Bacillus dNTP pool measurements were conducted as described (41). For each anthracis, Pseudomonas aeruginosa, S. cerevisiae, and Schizosac- measurement, three extract concentrations were used to ensure that the charomyces pombe has little or no effect on cell growth (12–16). extract did not inhibit the enzymatic reaction. The relative amounts of dNTPs

Nordman and Wright PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 10201 Downloaded by guest on September 24, 2021 Ϫ1 were calculated by: average cpm /␮l extract ϫ OD260 for WT and divided by in the presence of antibiotic selection. Once antibiotic selection is withdrawn, the value obtained of the relative extracts. pJN65 is readily lost because ndk is a nonessential gene, giving rise to blue/ white sectored colonies on LB plates supplemented with X-Gal. Synthetic NDP Kinase Assay. Overnight cultures were diluted 1:200 in 100 ml of LB and lethal interactions can be identiﬁed by looking for mutant cells unable to lose grown to OD600 Ϸ0.5. Cultures were rapidly chilled on an ice water bath and the plasmid and that form solid blue colonies on medium supplemented with centrifuged. Pellets were washed in10 mM Tris (pH 7.4), 10 mM MgCl2,10 mM X-Gal. Strain JN319/pJN65 was subjected to EZ::TN ϽKAN-2Ͼ transposon NaCl, 10 mM EDTA,and 1 mM DTT and stored at Ϫ80°C overnight. Pellets were mutagenesis according to the manufacturer’s recommendation (Epicentre) thawed on ice and resuspended in 2 ml of the above buffer, lysed by sonication, and plated on LB containing Kan and X-Gal. Candidate EZ-Tn5 insertions were Ϸ ϫ and centrifuged ( 16,000 g) to remove cell debris. Lysates were dialyzed sequenced by random PCR (43). overnight at 4°C in 10 mM Tris (pH 7.4), 10 mM NaCl, 10 mM MgCl2, 10 mM EDTA, and1 mM DTT by using 3,500 MW dialysis tubing (Spectra/Por). Extract protein ACKNOWLEDGMENTS. We thank Aaron Bernstein for conducting the ndk syn- concentration was determined by a Bradford assay. A pyruvate kinase/lactate thetic lethal screen; Jeffery H. Miller (University of California, Los Angeles), Andrei dehydrogenase-coupled assay was used to determine NDP kinase activity as Kuzminov (University of Illinois at Urbana-Champaign, Urbana, IL), Steve Sandler described (42) with CDP as the acceptor nucleoside diphosphate. (University of Massachusetts, Amherst, MA), and Jon Beckwith (Harvard Medical School, Boston) for contributing strains and plasmids used in this study; and Linc Synthetic Lethal Screen. pJN65 (lacϩ/ndkϩ) was introduced by transformation Sonenshein and Sergei Mirkin for critically reading the manuscript. This work was into a lac ndk strain of E. coli (JN319), rendering it phenotypically Lacϩ/Ndkϩ funded by a National Science Foundation grant (to A.W.).

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