title: 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 °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 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 °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 of datA sequence increases the relative amount of DNA synthesis of the nrdA101 mutant 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.
authors.affiliation: Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK.
authors.forename: Israel
authors.initials: I
authors.lastname: Salguero
authors.affiliation: Departmento de Bioquímica Biología Molecular y Genética, Universidad de Extremadura, 06071 Badajoz, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: López Acedo
authors.affiliation: Departmento de Bioquímica Biología Molecular y Genética, Universidad de Extremadura, 06071 Badajoz, Spain.
authors.forename: Elena C
authors.initials: EC
authors.lastname: Guzmán
mesh_terms: D000483:Alleles; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D005434:Flow Cytometry; D006358:Hot Temperature; D055550:Protein Stability; D012313:RNA / Q000096:biosynthesis; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics* / Q000378:metabolism*; D012293:Rifampin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D003854:Deoxyribonucleotides; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D012313:RNA; C449642:NrdA protein, E coli; D012262:Ribonucleoside Diphosphate Reductase; D012293:Rifampin