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

Synthetic lethality is inviability of a double-mutant combination of two fully viable single mutants, commonly interpreted as redundancy at an essential metabolic step. Thedut-1 defect inEscherichia 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 withdut 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 , andxthA ); (ii) double-strand DNA break repair (recA ,recBC , andruvABC ); and (iii) chromosomal-dimer resolution (xerC ,xerD , andftsK ). 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 anddegP ) 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 withdut can be explained by redundancy, by defect-damage-repair cycles, or as compensation.