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

Abstract

Cells carrying the thermosensitive nrdA101 allele are able to replicate entire chromosomes at 42°C when new DNA initiation events are inhibited. We investigated the role of the recombination enzymes on the progression of the DNA replication forks in the nrdA101 mutant at 42°C in the presence of rifampin. Using pulsed-field gel electrophoresis (PFGE), we demonstrated that the replication forks stalled and reversed during the replication progression under this restrictive condition. DNA labeling and flow cytometry experiments supported this finding as the deleterious effects found in the RecB-deficient background were suppressed specifically by the absence of RuvABC; however, this did not occur in a RecG-deficient background. Furthermore, we show that the RecA protein is absolutely required for DNA replication in the nrdA101 mutant at restrictive temperature when the replication forks are reversed. The detrimental effect of the recA deletion is not related to the chromosomal degradation caused by the absence of RecA. The inhibition of DNA replication observed in the nrdA101 recA mutant at 42°C in the presence of rifampin was reverted by the presence of the wild-type RecA protein expressed ectopically but only partially suppressed by the RecA protein with an S25P mutation [RecA(S25P)], deficient in the rescue of the stalled replication forks. We propose that RecA is required to maintain the integrity of the reversed forks in the nrdA101 mutant under certain restrictive conditions, supporting the relationship between DNA replication and recombination enzymes through the stabilization and repair of the stalled replication forks.Ribonucleotide reductase (RNR) is the only enzyme specifically required for the formation of deoxyribonucleotides (deoxynucleoside triphosphates [dNTP]), the precursors of DNA synthesis in Escherichia coli. RNR is a 1:1 complex of two nonidentical subunits, called R1 and R2, encoded by the genes nrdA and nrdB, respectively (8). The best-known defective RNR mutant in E. coli contains a thermolabile R1 subunit, coded by the nrdA101 allele. This allele carries a missense mutation, causing a change in amino acid 89 (L89P) (29). The activity of the enzyme measured in crude extracts of nrdA101 strains is limited to 6% of the wild type at 25°C (9), and the dNTP pool is lower than that of wild type even at the permissive temperature of 30°C (24). The RNR101 protein is inactivated in vitro after 2 min at 42°C (9) although a thermoresistant period of 50 min has been observed in vivo in the nrdA101 mutant strain (17). This thermoresistant period of DNA synthesis has been ascribed to the protection of RNR101 by the replication hyperstructure (15-17, 30, 31) and is extended when RNA and/or protein synthesis is inhibited, allowing cells to replicate entire chromosomes at 42°C (17). This feature has been related to a decrease in the overlap of replication rounds after the inhibition of new DNA initiation events (I. Salguero, E. López-Acedo, and E. C. Guzmán, submitted for publication).In a previous work we ...

Using a Chemical Genetic Screen to Enhance Our Understanding of the Antimicrobial Properties of Gallium against Escherichia coli

“…Another potential consequence of RNR inhibition is an increase in stalled replication forks, which are prone to DNA strand breakage [70]. Resumption of stalled replication forks and double strand breaks due to defective RNR function require the activity of recombination repair enzymes such as the RuvABC, RecBCD and RecA [71,72]. Our results support these observations since the deletion of recA , recD or ruvC triggered the Ga sensitive phenotype.…”

Template-switching during replication fork repair in bacteria

“…Michel and colleagues provided evidence for replication fork reversal and cleavage, using a combination of pulsed-field gel electrophoresis and genetic analysis of viability. They found evidence for fork reversal in a number of mutants defective in replication fork progression or after replication fork collision with strongly transcribed regions [40–44]; the laboratory of Guzman implicated replication fork reversal likewise under conditions of depleted deoxyribonucleotide pools [45, 46]. Under these various conditions, if the strain is defective in RecBCD nuclease activity, chromosomal cleavage is observed, dependent on the RuvC resolvase function.…”

Replication Fork Reversal after Replication–Transcription Collision

“…In several replication mutants, recombination proteins play a specific role by participating in a reaction named replication fork reversal (RFR) [5], [6], [19], [20], [21]. During RFR, the newly synthesized strands are unwound from the daughter duplexes and base pair to form a Holliday junction (HJ) adjacent to a DNA double-stranded (dsDNA) end.…”

“…In Escherichia coli , as in other organisms, recombination proteins have been shown to facilitate replication progression under various conditions of replication impairment [6] , [18] . In several replication mutants, recombination proteins play a specific role by participating in a reaction named replication fork reversal (RFR) [5] , [6] , [19] , [20] , [21] . During RFR, the newly synthesized strands are unwound from the daughter duplexes and base pair to form a Holliday junction (HJ) adjacent to a DNA double-stranded (dsDNA) end.…”