Defective Ribonucleoside Diphosphate Reductase Impairs Replication Fork Progression inEscherichia 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-g/ml) or under optimal (5-g/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.Ribonucleoside diphosphate reductase (NDP reductase) is the only specific enzyme required for the enzymatic formation of deoxyribonucleotides (dNTPs), the precursors of DNA synthesis in Escherichia coli. NDP reductase is a 1:1 complex of two nonidentical subunits called proteins R1 and R2, encoded by genes nrdA and nrdB, respectively (for a review, see reference 3). The best-known defective NDP reductase mutant of E. coli contains a thermolabile R1 subunit encoded by the nrdA101 allele. The activity of the enzyme measured in crude extracts of nrdA101 strains is limited to 6% of the wild-type activity at 25°C (6), and the dNTP pool is lower than wild type even at permissive temperatures (16). Our laboratory has shown that the presence of the nrdA101 allele lowers the replication rate of the mutant strain at the permissive temperature, as a nrdA101 mutant replicates the chromosome in 154 min at 30°C, while a nrdA ϩ strain does so in 98 min (10). Regarding the detrimental effect of the nrdA101 allele on the activity of the enzyme, this DNA replication effect is assumed to be due to the decrease in the NDP reductase activity as a dNTP provider. However, NDP reductase has been proposed as a component of the replication hyperstructure (10), and consequently the structure of the NDP reductase encoded by the nrdA101 allele might provoke a structural alteration of the replication hyperstructure that could also contribute to the lengthening of the C period in the mutant. A possible consequence of an altered replication hyperstr...

RecA-Dependent Replication in the nrdA101 (Ts) Mutant of Escherichia coli under Restrictive Conditions

“…In a previous work, we have demonstrated that the nrdA101 mutant undergoes RFR to restart the stalled replication forks (15). This suggested to us that RFR could be used to reactivate the DNA replication forks at 42°C in the presence of rifampin.…”

“…In a previous work we showed that in the nrdA101 mutant strain at 30°C, the DNA replication progresses with the help of replication fork reversal (RFR) (15). This mechanism has been extensively studied by Michel and coworkers (25).…”

Replication Restart in Bacteria

“…Both reactions result in the formation of a PriA substrate that allows replication restart. Replication fork reversal was observed (i) in several E. coli replication mutants in which different replisome components are affected (75), (ii) in the rep mutant that lacks the main helicase responsible for clearing DNA-bound proteins (mainly RNA polymerases) from the path of replication forks (76), (iii) in the priA mutant, suggesting that it occurs in cells that fail to restart replication after spontaneous arrest (77), (iv) in HU-treated cells and in a ribonucleotide reductase mutant (nrdA) (78,79), (v) after accumulation of topoisomerase I-DNA covalent complexes (80), (vi) in UV-irradiated cells (81), (vii) after encounter of replication forks with an oppositely oriented highly transcribed sequence (82), and (viii) in bacteria other than E. coli, such as in Pseudomonas syringae at low temperature (83).…”

Analysis of Chromosomal Replication Progression by Gel Electrophoresis

“…2C) (Seigneur et al, 1998). The occurrence of RFR at the stalled forks has been detected by this system in several replication mutants, such as in the helicase mutants rep and dnaBts (Michel et al, 1997;Seigneur et al, 2000), in holD G10 (Flores et al, 2001(Flores et al, , 2002, in dnaEts at 42ºC and in the dnaNts mutant at 37ºC (Grompone et al, 2002) and finally in nrdA101ts (Guarino et al, 2007a(Guarino et al, , 2007b. Fig.…”

“…2), it generates DSBs at arrested replication forks in recB deficient background (Seigneur et al, 1998); these results indicated the occurrence of replication fork reversal in nrdA101 mutant. As RFR is one of the mechanisms to restart the stalled replication forks, we could infer that the nrdA101 strain growing at 30°C increases the number of stalled replication forks that would proceed with the help of RFR process in a Rec+ proficient context (Guarino et al, 2007a).…”