Overlap of replication rounds disturbs the progression of replicating forks in a ribonucleotide reductase mutant of Escherichia coli

Abstract

Ribonucleotide reductase (RNR) is the only enzyme specifically required for the synthesis of deoxyribonucleotides (dNTPs). Surprisingly, Escherichia coli cells carrying the nrdA101 allele, which codes for a thermosensitive RNR101, are able to replicate entire chromosomes at 42 6C under RNA or protein synthesis inhibition. Here we show that the RNR101 protein is unstable at 42 6C and that its degradation under restrictive conditions is prevented by the presence of rifampicin. Nevertheless, the mere stability of the RNR protein at 42 6C cannot explain the completion of chromosomal DNA replication in the nrdA101 mutant. We found that inactivation of the DnaA protein by using several dnaAts alleles allows complete chromosome replication in the absence of rifampicin and suppresses the nucleoid segregation and cell division defects observed in the nrdA101 mutant at 42 6C. As both inactivation of the DnaA protein and inhibition of RNA synthesis block the occurrence of new DNA initiations, the consequent decrease in the number of forks per chromosome could be related to those effects. In support of this notion, we found that avoiding multifork replication rounds by the presence of moderate extra copies of datA sequence increases the relative amount of DNA synthesis of the nrdA101 mutant at 42 6C. We propose that a lower replication fork density results in an improvement of the progression of DNA replication, allowing replication of the entire chromosome at the restrictive temperature. The mechanism related to this effect is also discussed.

Relationship between Fork Progression and Initiation of Chromosome Replication in E. coli

“…After 4 h of incubation at 42ºC we found similar amount of DNA synthesis, either in the presence or in the absence of rifampicin at restrictive conditions. This result differs from that obtained in the nrdA101 mutant, whose DNA synthesis at 42ºC is half of the observed at 42 ºC when new initiations were inhibited (Table 2) (Salguero et al, 2011). As DNA initiation is not inhibited in the nrdA101/pMOR6 strain after the shift to 42ºC, none fully replicated chromosomes were detected at 42ºC (Fig.…”

“…We have verified that the number of overlapped replication forks per chromosome at 30ºC could be also lowered by introducing dnaA46, dnaA5 or dnaA508 alleles in the nrdA101 background (Table 2; Salguero et al, 2011). After incubation at 42ºC, all nrdA101 dnaAts strains exhibited a relative DNA synthesis and a thermoresistant period similar to those obtained by rifampicin addition either at 30ºC or 42ºC.…”

“…al., 2010); moreover, the nrdA gene is over-expressed in the presence of a defective DnaA protein (Gon et al, 2006, Lobner-Olesen et al, 2008. This over-expression is, however, unlikely to be responsible for the high residual DNA synthesis found in the nrdA101 strain at 42ºC since the chromosome is fully replicated at 42ºC in all studied nrdA101 dnaA strains even in the absence of protein synthesis (and hence absence of overproduced NrdA) (Salguero et al, 2011). In addition, over-expression of the nrdAB operon from a plasmid, leading to a doubling of enzyme activity as measured in cell-free extracts, causes only a doubling of the dATP, dCTP and dTTP pools without any increase in the dGTP pool .…”

“…It is intriguing that rifampicin or cloramphenicol addition, as well as the presence of a dnaAts allele, allowed the completion of chromosome replication in the nrdA101 mutant at the high temperature (Guzmán et al, 2002, Salguero et al, 2011. Inhibition of RNA and of protein synthesis, and inactivation of the DnaA protein all inhibit initiation of chromosome replication; therefore, completion of chromosome replication in the nrdA101 strain at 42ºC could be ascribed to the inhibition of new DNA initiations (Salguero et al, 2011). We suggest that the replication of the entire chromosome that occurs at the non-permissive temperature when new initiations are inhibited is due to a more efficient elongation as a consequence of the decreasing number of forks per chromosome.…”

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

“…The hda mutants that possess a defective negative regulator of replication initiation have an ori/ter ratio close to 5 (Morigen et al 2009). A defect in nucleotide reductase, the enzyme that supplies replication forks with DNA precursors, also increases the ori/ter ratio to 5 (Salguero et al 2011). A nucleotide reductase inhibitor, hydroxyurea (HU), reduces DNA replication rate in E. coli to 20% of the uninhibited rate (Kuong and Kuzminov 2009) and, within 4 hr, brings the ori/ter ratio to 5 (Kuong and Kuzminov 2012).…”