Vol. 134, No. 1

JOIJRNAL OF BACTERIOLOGY, Apr. 1978, p. 157-166

0021-9193/78/0134-0157$02.00/0 Copyright X 1978 American Society for Microbiology

Printed in U.S.A.

Escherichia coli Mutants Deficient in Deoxyuridine Triphosphatase STEVEN J. HOCHHAUSERt AND BERNARD WEISS* Department of Microbiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Received for publication 14 October 1977

Mutants deficient in deoxyuridine triphosphatase (dUTPase) were identified by enzyme assays of randomly chosen heavily mutagenized clones. Five mutants of independent origin were obtained. One mutant produced a thermolabile enzyme, and it was presumed to have a mutation in the structural gene for dUTPase, designated dut. The most deficient mutant had the following associated phenotypes: 98% congenic with the recipient is the least susceptible to error. parent, and the latter can then serve as a control DISCUSSION in biological experiments. Unfortunately, there Many of the phenotypic traits of dut mutants are no satisfactory techniques for the direct selection of dut markers in transductants. There- can be explained by the transient incorporation fore, we introduced into dut mutants markers of uracil into their DNA. A growing body of that are easily selectable in wild-type genetic evidence (22, 30a, 34) suggests that the major backgrounds and that are closely linked to dut. enzymes involved in the subsequent excision The markers were gltClO and TnlO. gltClO (for- repair are uracil-DNA glycosylase, exonuclease merly gltC5) specifies the constitutive overpro- III (containing the major endonuclease activduction of glutamate permease and enables the ity directed against apurinic/apyrimidinic cell to grow on glutamate as the sole carbon sites), DNA polymerase I, and DNA ligase. source (23). It was introduced into dut strains by Thus, e.g., the double mutants dutpoLA and dut P1 transduction with strain CS5 as the donor, 1g are each less viable than the corresponding and it was 20% cotransducible with pyrE and single mutants (30a), and dut xth (exonuclease dut. TnlO, a translocation element specifying III) double mutants display filamentous growth tetracycline resistance (16), was introduced into (A. Taylor and B. Weiss, unpublished data). In multiple loci in E. coli by transduction via a dut mutants, newly synthesized (pulse-labeled) nonintegrating A phage (X55b221cI8570am29- DNA strands are broken during the repair procTnlO) obtained from N. Kleckner. Tetr trans- ess, producing very short Okazaki fragments (Sof ductants were pooled, and a P1 lysate was pre- phenotype). Recombination, which involves pared from them. It was used to transduce a breakage and joining of DNA strands, is probapyrE strain to Pyr+ Tet' with simultaneous se- bly increased by this fragmentation, resulting in lection for both markers; recombinants, there- the hyper-Rec phenotype. The competition before, had TnlO insertions near pyrE. Three-fac- tween repair and replication for various subtor crosses indicated that in one such transduc- strates and enzymes might be responsible for the tant, TnlO was clockwise of and 70% cotrans- slow growth rate of dut-1 strains and, in part, for ducible with bothpyrE and dut. TnlO dut strains their reduced yields of phages P1 and 4X174. The increased frequency of spontaneous mutawere then constructed by P1 transduction. When gltClO dut strains were used as trans- tions in dut mutants might be explained by the ductional donors, selection was on minimal me- diversion of enzymes from the repair of other dium containing 0.5% sodium L-glutamate. After lesions, such as base mismatches resulting either incubation for 3 to 4 days at 370C, transductants from errors in replication or from the spontawere picked and restreaked to single colonies on neous deamination of DNA cytosine. Alternanutrient medium, rather than on selective me- tively, the fragmentation of DNA that accomdium, to save time. When TnlO dut strains were panies uracil excision might lead to the induction used as donors, transductions were carried out of an error-prone repair system (37), a hypothin the usual way, except that after adsorption of esis that might be tested with appropriate muphages, the cells were centrifuged, resuspended tants for that system. The mechanism of growth inhibition by uracil in nutrient broth containing 20 mM sodium citrate (to prevent reinfection), and aerated for 1 h is unknown. The exogenous uracil might be inat 370C to permit gene expression. Selection was corporated into DNA, or it might act through its on nutrient agar to which tetracycline powder known inhibition of uracil-DNA glycosylase

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(19). The latter possibility is less likely, however, because dut ung (glycosylase) double mutants are uracil resistant (B. Duncan, personal communication). Similarly, FUdR might act through its possibly increased incorporation into the DNA of dut mutants, or it might act through its inhibition of thymidylate synthetase (7), thereby decreasing the dTTP that would normally compete with dUTP for incorporation into DNA. The apparent incompatibility between pyrE and dut mutations is currently unexplainable. The pyrE gene codes for orotate phosphoribosyltransferase, an essential enzyme of pyridimidine metabolism. It is not clear why we failed to constructpyrE dut double mutants even though we used pyrimidine-containing media. dUTPase is required for the biosynthesis of most, if not all, of dTMP (4). About 75 to 80% of dUMP, its immediate precursor, comes from the pathway dCTP -* dUTP -- dUMP, and the rest comes from one or more other pathways including UDP -- dUDP -- dUTP -. dUMP. We might expect dut mutants to be thymine requiring, but they were not. Unfortunately, cells that were auxotrophic at 25°C were removed from the mutagenized population (24) before our screening for the dut mutants. To explore the possible essential nature of the dut gene in dTMP biosynthesis, we have recently attempted to produce very tight mutations by extensive deletions or insertions. We could not obtain insertions of Tn5 or TnlO elements into dut, nor could we obtain dut deletions promoted by nearby insertions of these elements. In our most intensive effort, we surveyed 15,000 Mucts (14) lysogens on a rich medium containing additional thymidine. We found 19 hyper-Rec colonies, none of which was dut. The effects of dut mutation are similar to those of thymine starvation. When thymineless E. coli are deprived of thymine, they lose viability, their DNA is broken, there is an increase in repair synthesis dependent onpolA, and there is an increase in mutational and recombinational frequencies (28). These effects are mimicked by the exposure of prototrophs to FUdR (8), an agent to which dut mutants are unusually sensitive. It is possible, therefore, that thymineless death may be mediated by the misincorporation of uracil into DNA resulting from a decreased production of dTTP. A fall in dTTP levels leads to derepression of dCTP deaminase (3). Large amounts of dUTP would then be produced and incorporated into DNA without competition from dTTP. The process by which E. coli makes most of its dTMP from triphosphate precursors is metabolically wasteful and entails the production of a harmful intermediate, dUTP, whose removal

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from DNA exacts an even higher metabolic cost. Perhaps dUTP is needed to serve an essential undiscovered function, perhaps some uracil misincorporation is desired because it promotes recombination and mutation and is thus beneficial to the ceil population, or perhaps dUTP production will make more sense when we come to understand the complex regulatory controls in pyrimidine biosynthesis (5). ACKNOWLEDGMENIS This research was supported by research grant NP 126 from the American Cancer Society and Public Health Service grant 1 P01 CA16519 from the National Cancer Institute. S.J.H. was supported by Public Health Service predoctoral training grant 5 T01 GM00184 from the National Institute of General Medical Sciences. We are grateful to Brian J. White for assistance with some of the experiments.

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Escherichia coli mutants deficient in deoxyuridine triphosphatase.

Vol. 134, No. 1 JOIJRNAL OF BACTERIOLOGY, Apr. 1978, p. 157-166 0021-9193/78/0134-0157$02.00/0 Copyright X 1978 American Society for Microbiology P...
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