Defective ribonucleoside diphosphate reductase impairs replication fork progression in Escherichia coli

Abstract

The observed lengthening of the C period in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in the nrdA101 mutant strain. We show here that the nrdA101 mutation induces DNA double-strand breaks at the permissive temperature in a recB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in the nrdA101 strain in a Rec(+) context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named "replication fork reversal." Viability results supported the occurrence of this process, as specific lethality was observed in the nrdA101 recB double mutant and was suppressed by the additional inactivation of ruvABC. None of these effects seem to be due to the limitation of the deoxyribonucleotide supply in the nrdA101 strain even at the permissive temperature, as we found the same level of DNA double-strand breaks in the nrdA(+) strain growing under limited (2-microg/ml) or under optimal (5-microg/ml) thymidine concentrations. We propose that the presence of an altered NDP reductase, as a component of the replication machinery, impairs the progression of the replication fork, contributing to the lengthening of the C period in the nrdA101 mutant at the permissive temperature.

FIG. 1.

The replication fork reversal model (adapted from reference with permission of the publisher). In the first step (A), the replication fork is arrested, causing fork reversal. The reversed fork forms an HJ (two alternative representations of this structure are shown, indicated by the open X and parallel stacked X). In Rec⁺ cells (B), RecBCD initiates RecA-dependent homologous recombination, and the resulting double HJ is resolved by RuvABC. Alternatively, if RecBCD encounters the HJ in the absence of RecA (C), the DNA double-strand end is degraded up to the HJ, restoring a fork structure. In both cases, replication restarts by a PriA-dependent process. In the absence of RecBCD (D), resolution of the HJ by RuvABC leads to DSBs at the stalled replication fork. Continuous line, parental chromosome; dashed lines, newly synthesized strands; disk, RuvAB; incised disk, RecBCD.

FIG. 2.

Representative profile of a PFGE experiment. JK626 (nrdA⁺ recB) (▴), JS628 (nrdA101 recB) (•), JK707 (nrdA⁺ recB ruvABC) (▵), and JS705 (nrdA101 recB ruvABC) (○) were used. Agarose plugs were prepared as described previously (20, 29). Gels were cut in 3-mm slices, the amount of [methyl-³H]thymidine present in each slice was measured, and the ratio of the total amount of [methyl-³H]thymidine in the lane was calculated for each slice. The gel origin is not shown; only the migrating DNA is shown. The position of the size marker is shown (Hansenula wingei; Bio-Rad). The amount of linear DNA was calculated from slice 4 to 12. Total proportions of migrating DNA in this experiment from slice 4 to 12 were 16.65% nrdA⁺ recB, 25.21% nrdA101 recB, 5.59% nrdA⁺ recB ruvABC, and 5.73% nrdA101 recB ruvABC.

FIG. 3.

DNA content per cell measured by flow cytometry after 4 h of incubation in the presence of rifampin and cephalexin in the nrdA⁺ strain growing at 30°C (A) and 37°C (B) and in the nrdA101 strain growing at 30°C (C) and 37°C (D).