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 colicells carrying thenrdA101allele, which codes for a thermosensitive RNR101, are able to replicate entire chromosomes at 42 °C under RNA or protein synthesis inhibition. Here we show that the RNR101 protein is unstable at 42 °C and that its degradation under restrictive conditions is prevented by the presence of rifampicin. Nevertheless, the mere stability of the RNR protein at 42 °C cannot explain the completion of chromosomal DNA replication in thenrdA101mutant. We found that inactivation of the DnaA protein by using severaldnaAtsalleles allows complete chromosome replication in the absence of rifampicin and suppresses the nucleoid segregation and cell division defects observed in thenrdA101mutant at 42 °C. 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 ofdatAsequence increases the relative amount of DNA synthesis of thenrdA101mutant at 42 °C. 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.