Defective ribonucleoside diphosphate reductase impairs replication fork progression in Escherichia coli.
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.
Guarino
Estrella
E
Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, 06080-Badajoz, Spain.
Jiménez-Sánchez
Alfonso
A
Guzmán
Elena C
EC
D002455:Cell Division / Q000235:genetics; D015169:Colony Count, Microbial; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics; D043211:Exodeoxyribonuclease V / Q000235:genetics; D045565:Holliday Junction Resolvases / Q000502:physiology; D050296:Microbial Viability; D009154:Mutation; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics* / Q000502:physiology*
D004269:DNA, Bacterial; D003854:Deoxyribonucleotides; D029968:Escherichia coli Proteins; D012262:Ribonucleoside Diphosphate Reductase; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D045565:Holliday Junction Resolvases