Synthetic Lethality with the dut Defect in Escherichia coli Reveals Layers of DNA Damage of Increasing Complexity Due to Uracil Incorporation

Abstract

Synthetic lethality is inviability of a double-mutant combination of two fully viable single mutants, commonly interpreted as redundancy at an essential metabolic step. The dut-1 defect in Escherichia coli inactivates dUTPase, causing increased uracil incorporation in DNA and known synthetic lethalities [SL(dut) mutations]. According to the redundancy logic, most of these SL(dut) mutations should affect nucleotide metabolism. After a systematic search for SL(dut) mutants, we did identify a single defect in the DNA precursor metabolism, inactivating thymidine kinase (tdk), that confirmed the redundancy explanation of synthetic lethality. However, we found that the bulk of mutations interacting genetically with dut are in DNA repair, revealing layers of damage of increasing complexity that uracil-DNA incorporation sends through the chromosomal metabolism. Thus, we isolated mutants in functions involved in (i) uracil-DNA excision (ung, polA, and xthA); (ii) doublestrand DNA break repair (recA, recBC, and ruvABC); and (iii) chromosomal-dimer resolution (xerC, xerD, and ftsK). These mutants in various DNA repair transactions cannot be redundant with dUTPase and instead reveal "defect-damage-repair" cycles linking unrelated metabolic pathways. In addition, two SL(dut) inserts (phoU and degP) identify functions that could act to support the weakened activity of the Dut-1 mutant enzyme, suggesting the "compensation" explanation for this synthetic lethality. We conclude that genetic interactions with dut can be explained by redundancy, by defect-damage-repair cycles, or as compensation.

The Genome-Wide Interaction Network of Nutrient Stress Genes in Escherichia coli

“…Deletion of either gene produces perfectly viable cells, while deletion of both is lethal. Other examples of synthetic lethality are found in various aspects of bacterial physiology such as DNA damage and repair (9), cell division (10), outer membrane biogenesis (11), and metabolism (12). It is worth noting that synthetic interactions often involve genes that are not linked on the chromosome and that are not related to each other.…”

Homologous Recombination—Experimental Systems, Analysis, and Significance

“…Starting with rdgB or dut mutants, that have elevated levels of spontaneous chromosomal fragmentation due to, correspondingly, hypoxanthine-DNA or uracil-DNA incorporation and excision, secondary mutations were isolated that made the original single mutants inviable (synthetically lethal with the rdgB or dut defects) (204, 324). Among such RdgB- or Dut-dependent mutants, there were multiple hits inactivating all the known six genes of recombinational repair of double-strand breaks in E. coli : recA , recB , recC , ruvA , ruvB and ruvC (204, 324). The prediction is that, if performed in the recBC sbcA or recBC sbcBC backgrounds, such a screen should yield all other known rec mutants, as well as (hopefully) some still unknown ones.…”

Deoxyuridine Triphosphate Incorporation during Somatic Hypermutation of Mouse VkOx Genes after Immunization with Phenyloxazolone

“…Uracil in DNA can arise by two mechanisms, cytidine deamination and misincorporation of dUTP. Both mechanisms are potentially mutagenic because of the formation of an AP during their repair (2)(3)(4), and it has been shown that a high intracellular concentration of dUTP is toxic in mammalian cells (5), yeast (6,7), and bacteria (8). It is interesting to note that most DNA polymerases do not discriminate dUTP from deoxythymidine triphosphate (dTTP), as shown by in vitro assays (3).…”