Lethality of a dut (deoxyuridine triphosphatase) mutation in Escherichia coli

Abstract

A chloramphenicol resistance gene was cloned into a plasmid-borne dut gene, producing an insertion mutation that was then transferred to the chromosome by allelic exchange. The mutation could not be acquired by haploid strains through substitutive recombination, even when two flanking markers were simultaneously transduced. The insertion was easily transferred, via generalized transduction, into the chromosomal dut region of strains harboring a A dut+ transducing phage; however, the resulting dut mutant/A, dut+ merodiploid could not then be cured of the prophage. This apparent lethality of the mutation could not be explained by effects on adjacent genes; the dfp gene retained complementing activity, and a ttk insertion mutant was viable.The dut gene product, deoxyuridine triphosphatase, is known to reduce incorporation of uracil into DNA and to be required in the de novo synthesis of thymidylate. Therefore, an attempt was made to determine whether the dut insertion would be tolerated in strains carrying the following compensatory mutations: dcd (dCTP deaminase) and cdd (deoxycytidine deaminase), which should reduce dUTP formation; ung (uracil-DNA glycosylase), which should reduce fatally excessive excision repair; deoA (thymidine phosphorylase), which should enhance the utilization of exogenous thymidine; and sulA, which should reduce the lethal side effects of SOS regulon induction. These mutations, either alone or in various combinations, did not permit the survival of a haploid dut insertion mutant, suggesting that the dut gene product might have an essential function apart from its deoxyuridine triphosphatase activity.

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

“…In E. coli, a considerable amount of dUTP is produced since it is an obligatory intermediate in the de novo synthesis of dTMP [23]. Although the DNA polymerases of E. coli do not effectively discriminate dUTP from dTTP, little dUTP finds its way into E. coli DNA because of the action of dUTP pyrophosphatase, which hydrolyzes dUTP to dUMP and PP i [10].…”

“…In early studies of dut mutants, it was anticipated that the dut mutant DNA would contain uracil [10]; however, the nascent DNA of these mutants was found to be exceptionally fragmented [3]. Such fragmentation was consistent with the excision of transiently incorporated uracil by uracil-DNA glycosylase (Ung) and subsequent incision of the Ung-generated abasic sites by apurinic/apyrimidinic endonuclease [23]. Inactivation of the ung gene permitted stable incorporation of uracil into the DNA of dut mutants [8].…”

“…CJ236 was constructed by C. Joyce (Yale University) from an isolate of BW313 (Hfr KL16 (PO-45) dut-1 ung-1 thi-1 relA1 spoT1; personal communication, B. Weiss, Emory University) that had spontaneously lost the Hfr phenotype by introducing a selectable F′ (pCJ105) [54]. Weiss and co-workers demonstrated that a dut null mutation was lethal, even in an ung background [23]; therefore, viable dut mutations are missense mutations. CJ236 was propagated at 25-30 °C in the presence of 125 μg/ml thymidine in order to avoid creation of secondary mutations, and checked for thymidine auxotrophy by plating at 42 °C as noted by el-Hajj et al [23] and B. Weiss (personal communication).…”

“…Weiss and co-workers demonstrated that a dut null mutation was lethal, even in an ung background [23]; therefore, viable dut mutations are missense mutations. CJ236 was propagated at 25-30 °C in the presence of 125 μg/ml thymidine in order to avoid creation of secondary mutations, and checked for thymidine auxotrophy by plating at 42 °C as noted by el-Hajj et al [23] and B. Weiss (personal communication). In order to identify the dut mutation(s) in CJ236, we amplified the dut gene in purified CJ236 genomic DNA using PCR and sequenced the PCR product as described under Section 2.…”

“…Subsequently, it was found that while some dut mutations were lethal, their lethality could be suppressed by null mutations in the dcd gene which, presumably, reduced the accumulation of dUTP and, hence, uracil in DNA [10]. Interestingly, dut insertion mutations (null mutations) were found to be lethal, regardless of compensatory mutations such as ung or dcd, suggesting that dUTP pyrophosphatase might serve a essential function apart from its nucleotidylhydrolase activity [23]. In Saccharomyces cerevisiae, the DUT1 gene is also required for viability [24].…”