JB Accepts, published online ahead of print on 23 May 2014 J. Bacteriol. doi:10.1128/JB.01718-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Genetic Requirements for Sensitivity of Bacteriophage T7 to Dideoxythymidine*

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Ngoc Q Tran, Stanley Tabor, and Charles C Richardson

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Department of Biological Chemistry and Molecular Pharmacology

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Harvard Medical School, Boston, Massachusetts 02115

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Running title: Thymidine Metabolism in Phage T7-infected E. coli

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To whom correspondence should be addressed: Department of Biological Chemistry and

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Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA

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02115. Tel: 617-432-1864; Fax: 617-432-3362; E-mail: [email protected]

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Key words: nucleotide kinase; thymine, thymidine; dideoxythymidine; DNA polymerase;

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DNA synthesis

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ABSTRACT

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We previously reported that the presence of dideoxythymidine (ddT) in the growth medium

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selectively inhibits the ability of bacteriophage T7 to infect Esherichia coli by inhibiting

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the phage DNA synthesis. In the presence of T7 gene 1.7 protein, ddT is taken up into the

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E. coli cell and converted to dideoxythymidine 5’-triphosphate (ddTTP). ddTTP is

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incorporated into DNA as ddTMP by the T7 DNA polymerase, resulting in chain

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termination. We have identified the pathway by which exogenous ddT is converted to

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ddTTP. The pathway consists of ddT transport by host nucleoside permeases and

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phosphorylation to ddTMP by the host thymidine kinase. T7 gene 1.7 protein

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phosphorylates ddTMP and ddTDP, resulting in ddTTP. A 74-residue peptide of the gene

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1.7 protein confers ddT sensitivity to the same extent as does the 196 residue wild-type

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gene 1.7 protein. We also show that cleavage of thymidine to thymine and deoxyribose-1-

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phosphate by the host thymidine phosphorylase greatly increases the sensitivity of phage

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T7 to ddT. Finally, a mutation in T7 DNA polymerase that leads to discrimination against

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the incorporation of ddTMP eliminates ddT sensitivity.

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INTRODUCTION

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When bacteriophage T7 infects an Escherichia coli cell, there is a rapid increase in

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DNA synthesis resulting in the production of over 100 T7 genomes in a 10 min period (1).

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The enzymes and mechanisms by which the T7 DNA is replicated have been studied in

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great detail. The T7 replisome consists of four proteins: T7 DNA polymerase, T7

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helicase/primase, T7 single-stranded DNA (ssDNA) binding protein, and E. coli

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thioredoxin (2). Much less well understood are the enzymes and mechanisms responsible

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for the production of the immediate precursors of DNA synthesis, the deoxynucleoside 5′-

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triphosphates (dNTPs). T7 derives most of the nucleotides found in its DNA from the

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breakdown of host DNA (3, 4). The host DNA is degraded to deoxynucleoside 5′-

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monophosphates (dNMPs) by the joint action of the gene 3 endonuclease and gene 6

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exonuclease (5, 6). How these dNMPs are eventually converted to dNTPs is not well

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understood. E. coli encodes four different dNMP kinases, each specific for one dNMP (7,

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8). At least one of these kinases, CMP kinase (CMK), is essential for T7 growth (9). It has

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generally been assumed that either the nucleoside diphosphate kinase (NDK) or the

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adenylate kinase (AMK) of the host converts the dNDPs to dNTPs (10) but the question

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remains as to whether the activity of these kinases is sufficient to meet the demand of T7

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DNA replication.

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In recent years we have reported a serendipitous finding that has led to new insight

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into nucleotide metabolism in T7-infected cells (11-13). Phage T7 growth and T7 DNA

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synthesis are inhibited by dideoxythymidine (ddT) at concentrations that are not toxic to E.

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coli. The inhibition of DNA synthesis suggests that the ddT is converted to ddTTP, and

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then the chain-terminating ddTMP is incorporated into T7 DNA by T7 DNA polymerase. 3

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T7 DNA polymerase incorporates ddNMPs with essentially the same efficiency as it does

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dNMPs (14). Therefore, we originally sought to screen for mutations in gene 5, the

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structural gene for T7 DNA polymerase (15), in order to identify those that would cause T7

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DNA polymerase to discriminate against the incorporation of ddNMPs. Surprisingly, when

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we isolated phage T7 that could grow in the presence of ddT, nearly all the mutations were

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in gene 1.7, a non-essential gene of unknown function. Extensive screening of mutants did

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identify a T7 phage that encodes an altered DNA polymerase, in which tyrosine 526 has

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been replaced by phenylalanine. This mutation bypasses the function of gene 1.7 and T7

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phage with this altered DNA polymerase are resistant to ddT (12). This single alteration in

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T7 DNA polymerase leads to discrimination against the incorporation of ddTMP by 8000-

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fold as compared with dTMP (14). These results strongly suggest that defects in gene 1.7

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lead to a defect in the conversion of ddT to ddTTP. Furthermore, overproduction of gp1.7

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from a plasmid renders E. coli cells sensitive to ddT, indicating that no other T7 proteins

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are required for conferring sensitivity to ddT. Both the inhibition of T7 phage and that of

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E. coli overproducing gp1.7 requires the E. coli thymidine kinase (12), suggesting that

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gp1.7 exerts its role after the formation of ddTMP.

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We have purified T7 gene 1.7 protein (gp1.7) and shown that it is indeed a

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nucleoside monophosphate kinase (11, 13). It has a number of remarkable properties that

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distinguish it from all other known nucleotide kinases. It phosphorylates dGMP and dTMP

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to dGDP and dTDP, respectively, using GTP, dGTP or dTTP as the phosphate donor. It

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phosphorylates ddTMP with an equal efficiency as dTMP, in contrast to the host

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thymidylate kinase (TMK), which discriminates over 500-fold against phosphorylation of

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ddTMP (11). This observation explains the phenotype of sensitivity to ddT resulting from

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the presence of gp1.7. T7 gp1.7 shares no sequence homology with any known protein,

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and there are no identifiable nucleotide binding motifs found in its protein sequence. A

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most unusual feature is its full activity as a kinase in the absence of any metal ion (11, 13).

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The identification of T7 gp1.7 as a nucleotide kinase would suggest that the E. coli

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nucleotide kinases are in fact not sufficient to provide an adequate supply of dNTPs for the

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synthesis of T7 DNA. dTTP in particular is required in large amounts by T7, not only as a

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substrate for DNA synthesis, but also as the energy supply for T7 DNA helicase where

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dTTP is hydrolyzed to dTDP (16). While the role of gp1.7 as a nucleoside monophosphate

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kinase has been clearly shown, there are other steps in the pathway from exogenous

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nucleosides in the media to the synthesis of dTTP precursors that are less understood. For

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example, in E. coli, thymidine is transported into the cell by the nup gene products that

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form a non-specific nucleoside permease (17). Upon entry into the cell, thymidine is either

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degraded into thymine and deoxyribose-1-phosphate by thymidine phosphorylase (18, 19),

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or it is phosphorylated to dTMP by thymidine kinase (Fig. 1). The sequestering of

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thymidine inside the cell requires its phosphorylation to dTMP since only the

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phosphorylated compound is unable to diffuse back out of the cell. Once thymidine is

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phosphorylated to dTMP, it can enter into the thymidine salvage pathway to be converted

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to dTTP. In fact, the thymidine salvage pathway is the only salvage pathway for

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deoxyribonucleosides in E. coli (20). Since phage T7 is no more sensitive to ddT in hosts

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that lack the tdk gene (12), the conversion of ddT to ddTMP must be catalyzed by the E.

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coli thymidine kinase, although it is not known whether any other E. coli genes may be

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required as well.

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The pathway by which ddTDP is phosphorylated to ddTTP in T7 phage-infected 5

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cells is not clear. It has generally been assumed that either the host nucleoside diphosphate

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kinase (NDK) or the adenylate kinase of the host converts the dNDPs to dNTPs (10) but the

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question remains whether the activity of these kinases also converts ddTDP to ddTTP.

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In this study, we use a genetic analysis to address these questions concerning the

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metabolism of thymidine and dideoxythymidine in E. coli infected with phage T7. A

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thorough understanding of this pathway will provide important detail how a bacteriophage,

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particularly phage T7, is selectively killed by ddT, as well as insight into the synthesis of

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the essential precursors used for both unwinding and synthesis of the DNA of replicating

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phage.

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MATERIALS AND METHODS Bacterial strain E. coli HMS89 (xth-1 thi argE mtl xyl rpsL ara his galK lacY proA leu

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thr tsx glnX44) was used for all experiments in which dideoxynucleosides were added to

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the media. E. coli HMS89 (DE3) was obtained by lyzogenizing E. coli HMS89 with the

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lambda phage DE3 using the λDE3 Lysogenization Kit (Novagen). This strain expresses

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T7 RNA polymerase upon induction by IPTG. E. coli DH5α (Invitrogen) was used for all

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sub-cloning manipulations. E. coli BL21 (DE3) (Novagen) was used for overexpression of

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gene 1.7. E. coli AGI containing the pCA24N-6His-tdk/tmk/ndk vector from the ASKA

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library was used for overexpression of the E. coli thymidine kinase, thymidylate kinase, and

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nucleoside diphosphate kinase as previously described (21). E. coli thymidine

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phosphorylase (Cat # T2807) was purchased from Sigma-Aldrich Co. T7 DNA

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polymerase/thioredoxin was purified and reconstituted as previous described (22).

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Radioactive materials were purchased from Moravek Biochemicals Inc.

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Purification of gp1.7 and nucleotide kinase assays. T7 gene 1.7 was cloned into the

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vector pET28a for overexpression in E. coli BL21 (DE3). The T7 gene 1.7 protein was

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purified as previously described (11). The standard nucleoside monophosphate kinase

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assay for gp1.7 measured the conversion of [3H]-d/ddTMP to [3H]-d/ddTDP. Reaction

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mixtures contained 100 mM Tris-HCl, pH 7.5, 5 mM DTT, 100 μM [H3]-d/ddTMP (~10

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cmp/pmole), 2.5 mM dTTP and 50 nM gp1.7. Reactions were carried out at 37 °C and

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were terminated by heating at 95 °C for 3 min. Where indicated the nucleoside

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diphosphate kinase activity of gp1.7 was measured by the conversion of [3H]-d/ddTDP to

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[3H]-d/ddTTP. Reaction products were analyzed by PEI Cellulose TLC as previously

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described (11).

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Thymidine kinase assay. Thymidine kinase activity was measured by the conversion

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of dT and ddT to dTMP and ddTMP, respectively. The reaction mixture (200 µl) contained

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200 μM [3H]-dT or [3H]-ddT (10 cpm/pmol), 5 mM ATP, 10 mM MgCl2, 100 mM Tris-

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HCl, pH 7.8, 20 μg BSA and 100 nM purified E. coli thymidine kinase. The reaction

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mixture was incubated at 37 °C. At indicated times, 20 µl aliquots were removed, and the

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reactions were terminated by heating the mixtures at 85 °C for 5 min. The samples were

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then applied to a PEI Cellulose Plate. The plate was wash thoroughly in distilled water and

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then air-dried. Chromatography was carried out in distilled water. The spot corresponding

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to dTMP was cut out and the radioactivity was measured using a liquid scintillation

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counter.

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Phosphorylase assay. E. coli thymidine phosphorylase catalyzes the phosphorolysis

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of thymidine to thymine and deoxyribose -1- phosphate. Phosphorolysis activity was

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assayed according to the method “Continuous Spectrophotometric Rate Determination”,

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described in detail elsewhere (23). The reaction mixture (1 ml) contained 200 mM

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potassium phosphate buffer, pH 7.4, 1 mM thymidine (or dideoxythymidine) and 10 units

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of thymidine phosphorylase. The decrease in A290 in a 1 cm light path cuvette was

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measured at room temperature using a Diode Array Spectrophotometer (Hewlett Packard).

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Construction of pDeoA and pTDK expression plasmids. The coding sequence of

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the deoA gene of E. coli was cloned into the vector pET28a. In the resulting plasmid

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pDeoA, the expression of the cloned deoA gene was under the control of a T7 RNA

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polymerase promoter. pTDK was constructed analogously. Plasmids were transformed

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into E. coli HMS89 strain for complementation studies.

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Inhibition of phage T7 by ddT and complementation assays. Inhibition of phage T7

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by ddT was carried out by mixing 300 μl of an overnight culture of E. coli HMS89 with 3

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ml of pre-melted soft agar (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.7% agar) at 45

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°C in the presence of 1 mM ddT. To the mixture, 50 μl of wild-type phage T7 (4 x 103 /ml)

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was added. The mixture was overlaid onto Petri plates. The plates were incubated in a 37

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°C incubator for six hours or left at room temperature (~25 °C) overnight. The plates were

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then photographed. Plating efficiencies were determined by dividing the number of

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plaques observed in the presence of ddT by the number observed in its absence.

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For mapping of the region of gp1.7 that is essential for ddT-sensitivity, gene 1.7

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mutants containing various deletions at either its 5’ or 3’ ends (see Fig. 11) were cloned

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into the expression vector pET28a. The resulting plasmids were then transformed into E.

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coli HMS89 for complementation analysis. In this system the gene 1.7 deletions will be

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expressed upon infection of the E. coli by phage T7. The cells are not infected by phag T7

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will grow normally in the presence of ddT. Complementation assays were carried out to

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determine the ability of the various truncated gp1.7 mutants to confer sensitivity of phage

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T7∆1.7 to the presence of ddT in the media. Phage T7∆1.7 has the entire gene 1.7 deleted

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and, in contrast to wild-type T7, is resistant to the presence of ddT in the media (1).

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Construction of gene knock-outs in E. coli HMS89. E. coli HMS89 mutants were

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constructed in which either deoA, deoB, deoC, deoD, dcd, ndk, nup, tdk or yjjG were

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deleted based on the protocol described in the Quick & Easy E. coli Gene Deletion Kit

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(Gene Bridges GmbH) and as previously described (12). The requirement of the deleted

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gene for conferring sensitivity of phage T7 to ddT in the media was examined by

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measuring the plating efficiency and plaque size of wild type phage T7 infecting each of

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the mutant E. coli strains in the presence of 1 mM ddT in the media, as described above.

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Preparation of E. coli crude lysate. E. coli HMS89 were grown in 10 ml LB at 37 °C

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to A600 = 1. Cells were harvested by centrifugation and then suspended in 500 μl of lysis

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buffer containing 20 mM Tris-HCl, pH 7.5, 10 mM β-mercaptoethanol and 0.1 mM EDTA.

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Cells were disrupted using a Branson Digital Sonifier at a setting of 20% pulse for 10 x 5

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sec, followed by centrifugation at 14,000 rpm for 30 min in a micro centrifuge. The

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supernatant was collected. The concentration of crude lysate was determined by the

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Bradford method (Bradford, 1976).

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Measurement of dideoxythymidine uptake. E. coli HMS89 tdk (DE3), and HMS89

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tdk (DE3)/pGP1.7 were grown at 30 °C to A600 = 0.3 in LB medium. Gene 1.7 was induced

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by the addition of 1 mM IPTG for 20 min. Nalidixic acid was added to a final

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concentration of 200 µg/ml and the cells were incubated for an additional 10 min to inhibit

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DNA synthesis. At time zero, [3H]-ddT was added to a final concentration of 100 μM (50

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μCi/ml). At the indicated times, 200 μl was removed, filtered by vacuum through nylon

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Millipore filters (pore size 0.45 μm), washed twice with 2 ml of cold LB medium, dried,

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and then the [3H] retained on the filters was measured using a liquid scintillation counter.

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Inhibition of DNA synthesis by dideoxythymidine in vitro. Kinase reaction mixtures

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(500 μl) contained 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 5 mM DTT, 0.5

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mM ddT, 2.5 mM ATP and 100 nM E. coli thymidine kinase. Reaction mixtures were pre-

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incubated at 37 °C for 30 min. DNA polymerase activity was then assayed as previously

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described (13). Briefly, DNA synthesis was initiated by the addition of the following

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components to the kinase mixture: 100 μM each dATP, dCTP, dGTP and [3H]-dTTP (~10

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cpm/pmole), 20 nM of single-stranded M13 DNA primed with a 24 nucleotide primer, and

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5 nM T7 DNA polymerase/thioredoxin. Where indicated, the reaction mixtures also

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contained 50 nM of either T7 gp1.7 or E. coli thymidylate kinase. DNA synthesis was

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carried out at 37 °C. Aliquots were removed at the indicated times and were spotted on

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DE81 ion exchange filters. Unincorporated nucleotides were removed by washing the

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filters three times (5 min each) in 300 mM ammonium formate. The [3H] retained on the

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filters was determined by liquid scintillation counting.

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RESULTS Effect of mutations in nucleoside uptake genes on the ability of dideoxythymidine

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to inhibit T7 phage growth. The uptake of nucleosides from the media into E. coli cells

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are believed to involve four nucleoside permeases, the products of the nupA (24), nupC

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(25), nupG (26) and nupX (27) genes. We tested whether any of these four gene products

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are solely responsible for the uptake of ddT into the cell by constructing different E. coli

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strains with a deletion of each of these genes. When T7 phage infected each of these

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strains, in each case the phage were inhibited to the same extent as in wild-type E. coli (Fig.

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2). Furthermore, we constructed strains containing deletions in combinations of two of the

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nup genes; T7 phage had the same sensitivity to ddT in these strains as well.

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Unfortunately, we were unsuccessful in our attempt to simultaneously knock out threes or

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four nup genes. The inability to do so could be due to technical difficulties, since at least

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three marker genes plus one from the plasmid must be inserted for selection. The results

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suggest that transport of ddT does not solely depend on a single or combination of two nup

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gene products.

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Effect of gene 1.7 on dideoxythymidine uptake. We previously showed that when

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T7 gp1.7 was overproduced in E. coli it dramatically increased the intracellular pools of

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dideoxythymidine nucleotides (ddTMP, ddTDP, and ddTTP) when the cells were grown in

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the presence of ddT in the media (11). However, this experiment could not distinguish

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between an effect of gp1.7 on the transport of ddT into the cell directly, or an indirect effect

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of sequestering the phosphorylated derivatives inside the cell as a result of their

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phosphorylated derivatives being unable to be transported out of the cell. In order to

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address this issue more directly, we examined the effect of gp1.7 on ddT transport using E.

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coli tdk. Since this strain lacks thymidine kinase activity which is required for the initial

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phosphorylation of ddT this experiment examines the role of T7 gp1.7 on ddT transport

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under conditions where the ddT will not be chemically altered when it enters the cell.

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Interestingly, gp1.7 significantly increases the intracellular concentration of ddT in this

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strain even though the nucleoside is not a substrate for gp1.7 (Fig. 3). The mechanism by

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which gp1.7 is facilitating this sequestration is not known.

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E. coli gene tdk is essential for conversion of ddT to ddTMP. The tdk gene of E.

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coli encodes for thymidine kinase (TDK, EC 2.7.1.21). Thymidine kinase plays an

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essential role in the thymidine salvage pathway by converting thymidine to thymidylate

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(dTMP) (Fig. 1). We have shown that phage T7 was able to grow on E. coli HMS89 tdk

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lacking the entire gene tdk in the presence of ddT up to 1.5 mM, with essentially no effect

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on the plating efficiency; the plaque size was comparable to those observed in the absence

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of ddT (12). In this study we purified thymidine kinase of E. coli and directly compared its

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ability to phosphorylate dT and ddT. E. coli thymidine kinase phosphorylates ddT to yield

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ddTMP, albeit at a rate ~20% that of dT to dTMP. Taken together these results show that

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E. coli thymidine kinase is the enzyme responsible for the conversion of ddT to ddTMP

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when T7 phage infects E. coli in the presence of ddT.

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Effect of host thymidine phosphorylase activity on ddT-sensitivity of phage T7.

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Gene deoA of E. coli encodes a thymidine phosphorylase (TP, EC 2.4.2.4) that catalyzes

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the reversible reaction: thymidine + phosphate  thymine + deoxyribose-1-phosphate (dR-

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1-P) (Fig. 1) (28, 29). Once thymidine is degraded to thymine and deoxyribose-1-

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phosphate, the thymidine is no longer incorporated into DNA since the conversion of

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thymine back to thymidine is inefficient except in thymine-requiring mutant strains.

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Therefore, an E. coli mutant that lacks thymidine phosphorylase effectively incorporates

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thymidine into DNA (30), and provides a useful tool for the measurement of DNA

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synthesis using labeled thymidine (30-32). We constructed the strain E. coli deoA

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containing a deletion of the entire deoA gene. We expected that T7 phage would be more

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sensitive to ddT in this strain, since the ddT would not be broken down to thymine and

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dideoxyribose-1-phosphate. Surprisingly, we found that in fact phage T7 was considerably

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less sensitive to ddT in E. coli deoA than in wild-type E. coli (Fig. 4A). Although phage T7

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produced smaller plaques when phage T7 infects E. coli deoA in the presence of ddT as

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compared with those produced in the absence of ddT, the efficiency of plating is essentially

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identical in both conditions (compare two upper plates, Fig. 4A). This effect is due to the

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absence of the deoA gene, since when the phage T7 infects E. coli deoA /pDeoA, which

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contains a plasmid that expresses the deoA gene upon infection of phage T7, they have the

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same high sensitivity to ddT as when the phage infect wild-type E. coli (lower right, Fig.

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4A).

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These results suggest that thymidine phosphorylase is required in order to obtain the

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maximum inhibition of phage T7 by ddT. This finding is contrary to what would be

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expected if thymidine phosphorylase cleaved ddT to thymine and ddR-1-P, comparable to

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the reaction it carries out with dT.

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To address this question, we compared the activities of purified E. coli thymidine

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phosphorylase on dT and ddT substrates to determine if a difference in activity could

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explain the effect of the presence of thymidine phosphorylase on the ability of ddT to

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inhibit T7 phage. We measured thymidine phosphorylase activity using a

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spectrophotometric assay based on the observation that dT and ddT have a higher molar

278

extinction coefficients than does thymine (see Materials and Methods). Cleavage of dT or 14

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ddT to produce thymine and dR-1-P or ddR-1-P will result in a decrease in the A290. When

280

dT is used as the substrate for thymidine phosphorylase there is a linear decrease of ~10%

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in A290 over 5 mins (left panel, Fig. 4B). In contrast, when ddT is the substrate for

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thymidine phosphorylase there is no detectable change in the A290 (right panel, Fig. 4B).

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We conclude that ddT is not a substrate of E. coli thymidine phosphorylase. This result

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explains why inhibition of phage T7 growth by ddT is significantly increased in the

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presence of thymidine phosphorylase. Thymidine phosphorylase will selectively degrade

286

dT over ddT, increasing the ratio of ddT to dT in the cell. A higher ratio of ddT to dT as a

287

substrate for thymidine kinase will result in a higher amount of ddTMP produced relative to

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dTMP.

289

T7 gp1.7, and not host enzymes, is responsible for the conversion of ddTDP to

290

ddTTP. In E. coli, nucleoside diphosphate kinase (NDK, EC 2.7.4.6) encoded by the ndk

291

gene is an ubiquitous enzyme responsible for the conversion of nucleoside diphosphates to

292

nucleoside triphosphates (33). The function of nucleoside diphosphate kinase was believed

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to be the maintenance of balanced nucleoside triphosphate pools in the cells (34, 35).

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However, the deletion of the ndk gene does not affect the viability of E. coli. Although we

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have previously shown (13) that gp1.7 can catalyze the synthesis of dTTP or ddTTP from

296

dTDP or ddTDP, respectively, we were curious as to the ability of nucleoside diphosphate

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kinase to convert ddTDP to ddTTP. We engineered E. coli ndk, and used this host to

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examine whether nucleoside diphosphate kinase was required to confer the sensitivity of

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phage T7 to ddT (Fig. 5). Serving as the controls, top plates of Fig. 5 show the plaques

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produced by T7 phage on wild-type E. coli in the absence and presence of 1 mM ddT,

301

respectively. Surprisingly, we obtained the same results when the E. coli strain is missing

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the ndk gene (lower plates, Fig. 5). Phage T7 produces plaques of normal size and

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frequency in the absence of ddT (compare the plates on left, Fig. 5), but is inhibited to the

304

same extent in the presence of ddT (compare plates on right, Fig. 5). This finding suggests

305

that the conversion of ddTDP to ddTTP is not dependent on nucleoside diphosphate kinase,

306

and there must be another enzyme responsible for this step.

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Lu and Inouye (10) have demonstrated that adenylate kinase (AMK, EC 2.7.4.3), an

308

essential enzyme for the generation of ADP from AMP, also has nucleoside diphosphate

309

kinase activity, and that this is the enzyme responsible for catalyzing this essential activity

310

in E. coli deleted for the ndk gene. In order to address the enzyme(s) responsible for

311

phosphorylation of ddTDP to ddTTP, we examined a reaction mixture containing either T7

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gp1.7 alone (solid lines, Fig. 6) or in the presence of a crude extract prepared from wild-

313

type E. coli (broken lines, Fig. 6). We followed the radioactive products produced starting

314

with either [H3]dTMP (Fig. 6A) or [H3]ddTMP (Fig. 6B) as substrates. Under these

315

conditions, T7 gp1.7 alone efficiently phosphorylates these substrates to [H3]dTDP (top

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solid line, Fig. 6A) and [H3]ddTDP (top solid line, Fig. 6B), respectively. There is also a

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small (~10%) amount of [H3]dTTP (bottom solid line, Fig. 6A) or [H3]ddTTP (bottom solid

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line, Fig. 6B). When the reaction mixtures contain the E. coli extract in addition to T7

319

gp1.7, the amount of each product formed is dramatically different depending on whether

320

the substrate is [H3]dTMP or [H3]ddTMP. When the substrate is [H3]dTMP, the major

321

product formed is [H3]dTTP (top broken line, Fig. 6A). However, when the substrate is

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[H3]ddTMP, the major product formed is [H3]ddTDP (top broken line, Fig. 6B). In fact,

323

the addition of the E. coli extract does not alter the amount of [H3]ddTTP formed as

324

compared to the small (~10%) amount formed in the presence of T7 gp1.7 alone (compare

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the bottom broken line to the bottom solid line, Fig. 6B). This demonstrates that while the

326

host enzymes efficiently phosphorylate dTDP to dTTP, it appears that there are no enzymes

327

in an E. coli lysate that efficiently phosphorylate ddTDP to ddTTP.

328

How does the gp1.7 phosphorylate both ddTMP and ddTDP? We have previously

329

shown that the thymidine kinase reaction catalyzed by T7 gp1.7 is reversible: ddTMP +

330

dTTP  dTDP + ddTDP (13). The reaction provides a pathway for the formation of

331

ddTTP via the reverse reaction involving two ddTDP substrates, or dTDP as a phosphate

332

donor.

333

Inhibition of DNA synthesis by ddT in vitro. Genetic and biochemical data have thus

334

far implicated the host tdk, T7 gene 1.7, and T7 gene 5 as essential genes for conferring

335

sensitivity of phage T7 to ddT in vivo. We purified these enzymes and reconstituted in

336

vitro the inhibition of DNA synthesis by ddT (Fig. 7). DNA synthesis reactions were

337

carried out coupled to the pre-incubated ddT-kinase reactions that contain E. coli thymidine

338

kinase and ddT and ATP as described in the Materials and Methods. DNA synthesis is

339

initiated by adding of T7 DNA polymerase, primed M13 ssDNA, and dNTPs. As shown in

340

Fig. 7, the addition of thymidylate kinase to the T7 DNA polymerase reaction has no effect

341

on the amount of DNA synthesized; under the conditions shown in Fig. 7, DNA synthesis is

342

linear for 90 min. However, when T7 gp1.7 was added to the reaction mixture, DNA

343

synthesis was strongly inhibited after approximately 30 min. These results show that these

344

three enzymes alone are sufficient to catalyze the synthesis of ddTTP from dT which then

345

is incorporated by T7 DNA polymerase to terminate DNA synthesis.

346 347

Mapping the region of gp1.7 that is essential for ddT-sensitivity. We previously analyzed a series of deletion mutant phage T7 to determine the region responsible for

17

348

conferring sensitivity of phage T7 to ddT (12). These results narrowed the critical region

349

for this activity to the C-terminal 113 residues of gp1.7. We have more precisely defined

350

the region essential for this activity via a series of additional deletions in gene 1.7 (Fig. 8).

351

Specific deletions were constructed in the plasmid pET28a, and then examined for their

352

ability to complement gp1.7 for the ability to confer ddT sensitivity to phage T7∆1.7 (Fig.

353

8A, see also in Materials and Methods). When 121 amino acid residues were deleted from

354

the N terminus of the T7 gp1.7 (gp1.7∆121N), the truncated protein was able to confer

355

nearly full sensitivity of the T7 phage to ddT (Fig. 8B). However, when one additional

356

amino acid residue was deleted from the N terminus (gp1.7∆122N), it was unable to

357

complement for gp1.7 and the T7∆1.7 phage was completely resistant to ddT.

358

We carried out a comparable analysis of deletions at the C terminus of gp1.7. Deletion

359

of the terminal residue at the C terminus (gp1.7∆1C) did not affect the ability of gp1.7 to

360

confer sensitivity of T7 phage to ddT (Fig. 8B). However, deletion of two residues

361

(gp1.7∆2C) resulted in complete loss of the ability of gp1.7 to confer sensitivity to ddT.

362

These results define the essential region of gp1.7 necessary for this activity to 74 residues at

363

the C-terminus of gp1.7 (Fig. 8A). It is remarkable that this truncated gp1.7, consisting of

364

just 38% of the wild-type gp1.7, is able to confer complete sensitivity of T7 phage to ddT.

365

18

366

DISCUSSION

367

Phage T7 growth is inhibited by dideoxythymidine (ddT) when it is added to the

368

growth media at concentrations that do not inhibit its host, E. coli (12). The inhibition is

369

the consequence of ddT being converted to ddTTP, which is then incorporated into T7

370

DNA as ddTMP to terminate further DNA synthesis (36). ddT must be taken up and

371

phosphorylated to ddTMP prior to being converted to ddTDP by gp1.7. Genetic and

372

biochemical data show that gp1.7 does not play a role in this step. In E. coli, thymidine is

373

phosphorylated to thymidylate (dTMP) by thymidine kinase (37). We previously showed

374

that when T7 infects an E. coli host lacking the tdk gene it is no longer inhibited by the

375

presence of ddT (12). This observation indicates that T7 phage does not encode a

376

thymidine kinase and that the host thymidine kinase is responsible for the conversion of

377

ddT to ddTMP. In this study we demonstrate that E. coli thymidine kinase indeed

378

phosphorylates ddT to ddTMP at a rate approximately 20% that of the rate it converts dT to

379

dTMP.

380

In this study we also examined the role of different E. coli genes on the uptake of ddT

381

into the cells. Four genes, nupA (24), nupC (25), nupG (26), and nupX (27), have been

382

shown to be responsible for transporting nucleosides into the cells. We show that deletion

383

of any of these four genes has no effect on the inhibition of T7 phage by ddT, suggesting

384

that none of these genes are solely responsible for uptake of ddT. T7 phage are also

385

inhibited normally by ddT in an E. coli mutant lacking two of the nup genes. However we

386

could not rule our one possibility is that each individual nup gene is able to transport ddT

387

into the cells. On the other hand, we have shown here that gp1.7 may play a role in ddT

388

uptake; when gp1.7 is produced in E. coli tdk, there is a significant increase in the uptake of

389

ddT. When E. coli cells lack the tdk gene, nucleosides taken up into the cell are unable to 19

390

be phosphorylated, and thus are free to pass back out of the cell. We do not know whether

391

gp1.7 plays a direct role on the transport of ddT or an indirect role by stimulating a host

392

gene product.

393

When dT is taken up by E. coli it can either be phosphorylated to dTMP by thymidine

394

kinase or it can be broken down by thymidine phosphorylase in the following reaction: dT

395

+ Pi  T + dR-1-P (29). While this reaction is reversible, in E. coli the breakdown of dT is

396

favored because thymidine phosphorylase is induced by thymidine and the fact that

397

thymine is not readily converted to dTTP for DNA synthesis. Breakdown of thymidine is

398

also favored by the metabolism of the resulting deoxyribose-1-P. At the outset of this study

399

we assumed that inactivation of the host thymidine phosphorylase would render T7 phage

400

more sensitive to ddT, since it would prevent its breakdown to deoxythymine and dR-1-P.

401

To our surprise, this mutation had the opposite effect, rendering phage T7 less sensitive to

402

ddT. This apparent paradoxical result was explained by our biochemical analysis of the

403

substrate requirements for the E. coli thymidine phosphorylase. While this enzyme readily

404

cleaves thymidine to thymine and dR-1-P, it was inactive with ddT. The consequence of

405

this differential activity results in increasing the relative intracellular concentration of ddT

406

versus dT by selectively degrading dT. Thus phage T7 is most sensitive to ddT when

407

thymidine phosphorylase is present to reduce the concentration of dT.

408

In E. coli, there are two pathways for the conversion of dNDPs to dNTPs, catalyzed by

409

nucleoside diphosphate kinase (33) and adenylate kinase (AMK) (10). In this study we

410

show that neither of these pathways are responsible for the conversion of ddTDP to ddTTP

411

in T7-infected cells. Nucleoside diphosphate kinase is a nonessential enzyme. Phage T7 is

412

fully inhibited by ddT when it infects E. coli mutants lacking this enzyme. Furthermore,

413

whereas wild-type E. coli extracts efficiently convert dTDP to dTTP, they are unable to 20

414

phosphorylate ddTDP to ddTTP. We have previous shown that T7 gp1.7 is a reversible

415

kinase, catalyzing the reaction d/ddTDP + ddTDP  d/ddTMP + ddTTP (13). When

416

labeled ddTMP is used as the substrate, ~10% of the ddTDP generated is consistently

417

converted to ddTTP. The amount of ddTTP generated is limited in these reactions since the

418

phosphate donor dTTP is always present in large excess, favoring the generation of

419

dTDP/ddTDP. In T7-infected E. coli cells, the dTTP concentration is much lower since it

420

is a substrate for both T7 DNA polymerase and T7 helicase, being hydrolyzed to dTDP to

421

drive unwinding activity. These conditions would favor the production of ddTTP.

422

Consistent with this scenario, we have shown previously that in vivo, gp1.7 dramatically

423

increases the ddTTP pool in E. coli cells growing in the presence of ddT (11). We

424

conclude that ddTDP is not a substrate for either of the two host pathways for the

425

conversion to the nucleoside triphosphate, and that in T7-infected cells gp1.7 is the enzyme

426

responsible for both the conversion of ddTMP to ddTDP and its subsequent

427

phosphorylation to ddTTP. It remains to be determined whether this is an important

428

pathway for the conversion of dTDP to dTTP as well, or if the host nucleoside diphosphate

429

kinase or AMP kinase pathways dominate with this substrate.

430

We believe that we have identified all the host and phage genes involved in ddT

431

metabolism. We did not see any difference in the sensitivity of phage T7 to ddT when

432

infecting E. coli mutants lacking the deoB, deoC, deoD, yjjG or dcd genes, mutations that

433

have been shown to increase the ability of E. coli to use exogenous thymidine (19, 38, 39).

434

Moreover, we could reconstitute in vitro the ddT inhibition of DNA synthesis catalyzed by

435

T7 DNA polymerase by the addition of the two nucleotide kinases: E. coli thymidylate

436

kinase and T7 gp1.7.

437

In addition to its ability to phosphorylate dideoxynucleotides, gp1.7 has a number of 21

438

other novel properties not found in other known kinases. For example, it does not share

439

any sequence homology with any other kinase, including any of the identifiable motifs

440

found in nucleotide binding domains (12). Furthermore, it lacks any requirement for a

441

divalent cation for catalytic activity (11). We had previously identified the region of gp1.7

442

essential for conferring ddT sensitivity to lie in the 113 carboxyl-terminal residues of the

443

protein. In this study, we more precisely mapped the residues essential for activity. The

444

ability to confer sensitivity to ddT was retained after deleting 121 residues at the amino

445

terminus and just a single residue from the carboxyl terminus, leaving a minimal fragment

446

of 74 residues that retained kinase activity. The role of the amino terminal 121 residues is

447

not known. This region has nine cysteine residues arranged in two putative zinc-binding

448

motifs. One possibility is that this region may play a role in physical interactions between

449

gp1.7 and other T7 DNA replication proteins.

450

22

451

ACKNOWLEDGEMENT

452

* This work was funded by U. S. Public Health Service Grant # GM54397 and Department

453

of Energy Grant DE-FG02-96ER 62251

454

To whom correspondence should be addressed: Department of Biological Chemistry and

455

Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA

456

02115. Tel: 617-432-1864; Fax: 617-432-3362; E-mail: [email protected]

457

Abbreviations: gp1.7, T7 nucleotide kinase; gp5, T7 DNA polymerase; TDK, thymidine

458

kinase; TMK, thymidylate kinase; NDP, nucleoside diphosphate kinase; TP, thymidine

459

phosphorylase; dT, thymidine; ddT, dideoxythymidine; dR-1-P, deoxyribose -1- phosphate;

460

ddNTPs, dideoxynucleoside 5’-triphosphates; TLC, thin layer chromatography.

461

23

462

REFERENCES

463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507

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Kruger DH, Schroeder C. 1981. Bacteriophage T3 and bacteriophage T7 virushost cell interactions. Microbiological reviews 45:9-51. Hamdan SM, Loparo JJ, Takahashi M, Richardson CC, van Oijen AM. 2009. Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature 457:336-339. Labaw LW. 1951. The Origin of Phosphorus in T5 Bacteriophage of Escherichia Coli. Proc Natl Acad Sci U S A 37:721-725. Labaw LW. 1953. The origin of phosphorus in the T1, T5, T6, and T7 bacteriophages of Escherichia coli. J Bacteriol 66:429-436. Center MS, Richardson CC. 1970. An endonuclease induced after infection of Escherichia coli with bacteriophage T7. I. Purification and properties of the enzyme. J Biol Chem 245:6285-6291. Kerr C, Sadowski PD. 1972. Gene 6 exonuclease of bacteriophage T7. I. Purification and properties of the enzyme. J Biol Chem 247:305-310. Neuhard J, Kelln RA. 1996. Biosynthesis and conversion of pyrimidines in Escherichia coli and Salmonella, p. 580-599, Cellular and Molecular Biology, 2nd ed ed, vol. 1. ASM Press, Washington DC. Zalkin H, and Nygaard, P. 1996. Biosynthesis of purine nucleotide in Escherichia coli and Salmonella: Cellular and Molecular Biology, p. 561-579., 2nd ed. ed, vol. 1. ASM Press, Washington DC. Qimron U, Marintcheva B, Tabor S, Richardson CC. 2006. Genomewide screens for Escherichia coli genes affecting growth of T7 bacteriophage. Proc Natl Acad Sci U S A 103:19039-19044. Lu Q, Inouye M. 1996. Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism. Proc Natl Acad Sci U S A 93:57205725. Tran NQ, Lee SJ, Richardson CC, Tabor S. 2010. A novel nucleotide kinase encoded by gene 1.7 of bacteriophage T7. Mol Microbiol 77:492-504. Tran NQ, Rezende LF, Qimron U, Richardson CC, Tabor S. 2008. Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine. Proc Natl Acad Sci U S A 105:9373-9378. Tran NQ, Tabor S, Amarasiriwardena CJ, Kulczyk AW, Richardson CC. 2012. Characterization of a nucleotide kinase encoded by bacteriophage T7. J Biol Chem 287:29468-29478. Tabor S, Richardson CC. 1995. A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc Natl Acad Sci U S A 92:6339-6343. Doublié S, Tabor S, Long A, Richardson CC, Ellenberger T. 1998. Crystal structure of bacteriophage T7 DNA polymerase complexed to a primer-template, a nucleoside triphosphate, and its processivity factor thioredoxin. Nature 391:8. Matson SW, Tabor S, Richardson CC. 1983. The gene 4 protein of bacteriophage T7. Characterization of helicase activity. J Biol Chem 258:14017-14024. McKeown M, Kahn M, Hanawalt P. 1976. Thymidine uptake and utilization in Escherichia coli: a new gene controlling nucleoside transport. J Bacteriol 126:814822. 24

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18. 19. 20. 21.

22. 23. 24. 25. 26. 27.

28. 29. 30. 31. 32. 33. 34. 35.

Ahmad SI, Kirk SH, Eisenstark A. 1998. Thymine metabolism and thymineless death in prokaryotes and eukaryotes. Annu Rev Microbiol 52:591-625. O'Donovan GA, Neuhard J. 1970. Pyrimidine metabolism in microorganisms. Bacteriol Rev 34:278-343. Karlstrom HO. 1970. Inability of Escherichia coli B to incorporate added deoxycytidine, deoxyandenosine, and deoxyguanosine into DNA. Eur J Biochem 17:68-71. Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H, Mori H. 2005. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 12:291-299. Tran NQ, Lee SJ, Akabayov B, Johnson DE, Richardson CC. 2012. Thioredoxin, the processivity factor, sequesters an exposed cysteine in the thumb domain of bacteriophage T7 DNA polymerase. J Biol Chem 287:39732-39741. Krenitsky TA, Bushby SRM. 1979. . United States Patent 4, 178,212, 1-8 Burroughs Wellcome Co., Research Triangle Park, NC. Maier C, Bremer E, Schmid A, Benz R. 1988. Pore-forming activity of the Tsx protein from the outer membrane of Escherichia coli. Demonstration of a nucleoside-specific binding site. J Biol Chem 263:2493-2499. Craig JE, Zhang Y, Gallagher MP. 1994. Cloning of the nupC gene of Escherichia coli encoding a nucleoside transport system, and identification of an adjacent insertion element, IS 186. Mol Microbiol 11:1159-1168. Westh Hansen SE, Jensen N, Munch-Petersen A. 1987. Studies on the sequence and structure of the Escherichia coli K-12 nupG gene, encoding a nucleosidetransport system. Eur J Biochem 168:385-391. Saier MH, Jr., Eng BH, Fard S, Garg J, Haggerty DA, Hutchinson WJ, Jack DL, Lai EC, Liu HJ, Nusinew DP, Omar AM, Pao SS, Paulsen IT, Quan JA, Sliwinski M, Tseng TT, Wachi S, Young GB. 1999. Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim Biophys Acta 1422:1-56. Manson LA, Lampen JO. 1951. The metabolism of desoxyribose nucleosides in Escherichia coli. J Biol Chem 193:539-547. Schwartz M. 1971. Thymidine phosphorylase from Escherichia coli. Properties and kinetics. Eur J Biochem 21:191-198. Fangman WL, Novick A. 1966. Mutant bacteria showing efficient utilization of thymidine. J Bacteriol 91:2390-2391. Budman DR, Pardee AB. 1967. Thymidine and thymine incorporation into deoxyribonucleic acid: inhibition and repression by uridine of thymidine phosphorylase of Escherichia coli. J Bacteriol 94:1546-1550. Fangman WL. 1969. Specificity and efficiency of thymidine incorporation in Escherichia coli lacking thymidine phosphorylase. J Bacteriol 99:681-687. Parks RJ, Agarwal R. 1973. Nucleoside diphosphokinases. The Enzymes, ed Boyer PD (Academic, New York) 8:307-333. Almaula N, Lu Q, Delgado J, Belkin S, Inouye M. 1995. Nucleoside diphosphate kinase from Escherichia coli. J Bacteriol 177:2524-2529. Miller JH, Funchain P, Clendenin W, Huang T, Nguyen A, Wolff E, Yeung A, Chiang JH, Garibyan L, Slupska MM, Yang H. 2002. Escherichia coli strains 25

555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574

36. 37. 38. 39. 40. 41. 42.

(ndk) lacking nucleoside diphosphate kinase are powerful mutators for base substitutions and frameshifts in mismatch-repair-deficient strains. Genetics 162:513. Doering AM, Jansen M, Cohen SS. 1966. Polymer synthesis in killed bacteria: lethality of 2',3'-dideoxyadenosine. J Bacteriol 92:565-574. Okazaki R, Kornberg A. 1964. Deoxythymidine Kinase of Escherichia Coli. I. Purification and Some Properties of the Enzyme. J Biol Chem 239:269-274. O'Donovan GA, Edlin G, Fuchs JA, Neuhard J, Thomassen E. 1971. Deoxycytidine triphosphate deaminase: characterization of an Escherichia coli mutant deficient in the enzyme. J Bacteriol 105:666-672. Weiss B. 2007. YjjG, a dUMP phosphatase, is critical for thymine utilization by Escherichia coli K-12. J Bacteriol 189:2186-2189. Okazaki R, Kornberg A. 1964. Deoxythymidine Kinase of Escherichia Coli. Ii. Kinetics and Feedback Control. J Biol Chem 239:275-284. Reynes JP, Tiraby M, Baron M, Drocourt D, Tiraby G. 1996. Escherichia coli thymidylate kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk locus. J Bacteriol 178:2804-2812. Belfort M, Maley GF, Maley F. 1983. Characterization of the Escherichia coli thyA gene and its amplified thymidylate synthetase product. Proc Natl Acad Sci U S A 80:1858-1861.

575

26

576

FIGURE LEGENDS

577

FIG 1. Thymidine salvage pathway in E. coli. deoA, thymidine phosphorylase (28); tdk,

578

thymidine kinase (40); yjjG, dUMP phosphatase (39); tmk, thymidylate kinase (41); ndk,

579

nucleoside diphosphate kinase (33); amk, adenylate kinase (10); thyA, thymidylate synthase

580

(42); dT, thymidine; dU, deoxiuridine; dR-1-P, deoxyribose-1-phosphate; broken arrow,

581

synthesis de novo.

582

FIG 2. Role of E. coli nup genes in sensitivity of phage T7 to ddT in the media. For each

583

pair of plates shown, T7 phage was plated on a different E. coli strain either in the absence

584

of ddT (left) or the presence of 1 mM ddT (right). From top to bottom: E. coli HMS89 wt,

585

E. coli HMS89 nupA, E. coli HMS89 nupC, E. coli HMS89 nupG, and E. coli HMS89

586

nupX. Plates were incubated at 37 °C for 4 hr and then photographed.

587

FIG 3. Effect of gene 1.7 protein on dideoxythymidine uptake. E. coli strain HMS89

588

(DE3) tdk () or HMS89(DE3) tdk/ pGP1.7 () were grown at 37 °C to A600 = 0.4 in LB

589

medium. Cells were induced with 1 mM IPTG for 30 min followed by addition of 250

590

μg/ml nalixidic acid to inhibit DNA synthesis. [3H]ddT was then added to a final

591

concentration of 100 μM (50 μCi/ml). Aliquots of 200 μl were removed at the indicated

592

times and the [3H]ddT taken up by the cells was determined as described in “Materials and

593

Methods”.

594

FIG 4. Requirement of host thymidine phosphorylase (TP) for maximum inhibition of

595

phage T7 by ddT.

596

A. Effect of E. coli thymidine phosphorylase on ddT-sensitivity of phage T7. Top: E. coli

597

deoA infected with phage T7 in the absence (left) and in the presence of 1.5 mM ddT 27

598

(right). Bottom: E. coli deoA carrying the pDeoA plasmid infected with phage T7 in the

599

absence of ddT (left) and in the presence of 1.5 mM ddT. pDeoA will induce the

600

expression of the deoA gene upon infection of phage T7. Number of phage infected

601

HMS89 deoA, and HMS89 deoA/ pDeoA in the presence of ddT are 10 and 100-folds

602

higher than in the absence of ddT, respectively. Plates were left at room temperature over-

603

night and then photographed. Black circles indicate plaques of phage T7 formed on the

604

lawn of E. coli cells.

605

B. Comparison of the ability of E. coli thymidine phosphorylase to use thymidine and

606

dideoxythymidine. Reaction mixtures contained either thymidine (left panel) or

607

dideoxythymidine (right panel) and 10 units of enzyme thymidine phosphorylase in 100

608

mM KH2PO4, pH 7.4. The decrease in A290 in a 1 cm light path at room temperature was

609

monitored using a Diode Array Spectrophotometer (Hewlett Packard), as described under

610

“Materials and Methods” and elsewhere (23). () control reactions in the absence of

611

enzyme; () reactions contain 10 units of thymidine phosphorylase.

612

FIG 5. Host nucleoside diphosphate kinase is not required for gene 1.7-dependent ddT

613

sensitivity of phage T7. Upper panel: Wild-type E. coli HMS89 infected with phage T7 in

614

the absence (left) and presence (right) of 1 mM ddT. Lower panel: E. coli HMS89 ndk

615

infected with phage T7 in the absence (left) and presence (right) of 1 mM ddT. Plates were

616

incubated at 37 oC for 5 hrs then photographed. Black circles indicate plaques of phage T7

617

formed on the lawn of E. coli cells.

618

FIG 6. Roles of host enzymes in the conversion of ddTDP to ddTTP. A. Reaction

619

mixtures (100 μl) contained 100 μM [H3]-dTMP, 2.5 mM dTTP and ATP, and 10 mM

620

Mg2+. Reactions were initiated by the addition of 50 ng of gp1.7 (solid line) or 50 ng of 28

621

gp1.7 + 2.5 μg of E. coli crude lysate (broken line). B. An identical experiment in A was

622

performed except that [H3]-dTMP was replaced by [H3]-ddTMP. At the indicated times,

623

the formation of [3H]-dTDP () and [3H]-dTTP () (left panel) or [3H]-ddTDP () and

624

[3H]-ddTTP () (right panel) were determined by TLC as previously described (11).

625

FIG 7. Reconstitution of the inhibition of DNA synthesis by ddT in vitro. ddT kinase

626

reaction mixtures contained 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 5 mM

627

DTT, 0.5 mM ddT, 2.5 mM ATP and 100 nM E. coli thymidine kinase were incubated at

628

37 °C for 30 min. DNA synthesis assay was initiated by the addition of the following

629

components to the kinase mixture: 100 μM each dATP, dCTP, dGTP and [3H]-dTTP, 20

630

nM of M13 ssDNA primed with a 24 nucleotide primer, and 5 nM T7 DNA polymerase

631

(). Where indicated, the addition also contained 50 nM of either T7 gp1.7 () or E. coli

632

thymidylate kinase (). DNA synthesis was carried out at 37 °C. [H3]-dTMP incorporated

633

into DNA was measured as previous described (13).

634

FIG 8. Deletion mapping of the region of gp1.7 essential for conferring ddT-sensitivity to

635

T7 phage growth.

636

A. Schematic of deletion map. Horizontal bars: Δ125N and Δ126N, gp1.7 deletion mutants

637

missing the first 125 and 126 residues at N-terminus, respectively; Δ1C and Δ2C, gp1.7

638

deletion mutants missing the terminal 1 and 2 residues at the C-terminus, respectively.

639

DNA sequences encode truncated gp1.7 were cloned into expression plasmid pET28a. The

640

resulting plasmids were transformed into E. coli HMS89 for examining the ability of the

641

truncated gp1.7 to confer T7Δ1.7 sensitive to ddT as described in B. The column at the

642

right summarizes the effect of each deletion on the ability of the altered gp1.7 to confer

643

sensitivity of T7∆1.7 growth to ddT: (+), confers inhibition and (-) confers no inhibition. 29

644

B. Ability of truncated gp1.7 to confer T7∆1.7 sensitive to ddT. T7Δ1.7 (lacking the entire

645

gene 1.7) was used to infect E. coli HMS89 containing pGP1.7 mutant plasmids that

646

express the truncated forms of gp1.7 described in A. Complementation was performed as

647

described in “Materials and Methods”.

30