Catalytically inactive T7 DNA polymerase imposes a lethal replication roadblock.

Bacteriophage T7 encodes its own DNA polymerase, the product of gene 5 (gp5). In isolation, gp5 is a DNA polymerase of low processivity. However, gp5 becomes highly processive upon formation of a complex with thioredoxin, the product of the gene. Expression of a gp5 variant in which aspartate residues in the metal-binding site of the polymerase domain were replaced by alanine is highly toxic to cells. This toxicity depends on the presence of a functional allele and T7 RNA polymerase-driven expression but is independent of the exonuclease activity of gp5. , the purified gp5 variant is devoid of any detectable polymerase activity and inhibited DNA synthesis by the replisomes of and T7 in the presence of thioredoxin by forming a stable complex with DNA that prevents replication. On the other hand, the highly homologous Klenow fragment of DNA polymerase I containing an engineered gp5 thioredoxin-binding domain did not exhibit toxicity. We conclude that gp5 alleles encoding inactive polymerases, in combination with thioredoxin, could be useful as a shutoff mechanism in the design of a bacterial cell-growth system.