1887

Microbiology Society logo

Volume 157, Issue 7

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

Abstract

Ribonucleotide reductase (RNR) is the only enzyme specifically required for the synthesis of deoxyribonucleotides (dNTPs). Surprisingly, cells carrying the 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 mutant. We found that inactivation of the DnaA protein by using several alleles allows complete chromosome replication in the absence of rifampicin and suppresses the nucleoid segregation and cell division defects observed in the 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 sequence increases the relative amount of DNA synthesis of the 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.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Funding
This study was supported by the:
  • Junta de Extremadura (Award PRI07A001)
  • Ministerio de Educación y Ciencia (Award BFU2007-63942)
  • Ministerio de Educación y Cultura (Award FPU AP2002-1157)
© 2011 SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.047316-0
2011-07-01
2024-04-25
Loading full text...

Full text loading...

/deliver/fulltext/micro/157/7/1955.html?itemId=/content/journal/micro/10.1099/mic.0.047316-0&mimeType=html&fmt=ahah

References

  1. Atlung T., Hansen F. G. ( 1993). Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli . J Bacteriol 175:6537–6545[PubMed]
     [Google Scholar]
  2. Dix D. E., Helmstetter C. E. ( 1973). Coupling between chromosome completion and cell division in Escherichia coli . J Bacteriol 115:786–795[PubMed]
     [Google Scholar]
  3. Eliasson A., Nordström K., Bernander R. ( 1996). Escherichia coli strains in which chromosome replication is controlled by a P1 or F replicon integrated into oriC . Mol Microbiol 20:1013–1023 [View Article][PubMed]
     [Google Scholar]
  4. Fuchs J. A., Karlström H. O., Warner H. R., Reichard P. ( 1972). Defective gene product in dnaF mutant of Escherichia coli . Nat New Biol 238:69–71[PubMed] [CrossRef]
     [Google Scholar]
  5. Gibert I., Calero S., Barbé J. ( 1990). Measurement of in vivo expression of nrdA and nrdB genes of Escherichia coli by using lacZ gene fusions. Mol Gen Genet 220:400–408 [View Article][PubMed]
     [Google Scholar]
  6. Gon S., Camara J. E., Klungsøyr H. K., Crooke E., Skarstad K., Beckwith J. ( 2006). A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli . EMBO J 25:1137–1147 [View Article][PubMed]
     [Google Scholar]
  7. Gottesman S. ( 2003). Proteolysis in bacterial regulatory circuits. Annu Rev Cell Dev Biol 19:565–587 [View Article][PubMed]
     [Google Scholar]
  8. Gottesman S., Wickner S., Maurizi M. R. ( 1997). Protein quality control: triage by chaperones and proteases. Genes Dev 11:815–823 [View Article][PubMed]
     [Google Scholar]
  9. Grigorian A. V., Lustig R. B., Guzmán E. C., Mahaffy J. M., Zyskind J. W. ( 2003). Escherichia coli cells with increased levels of DnaA and deficient in recombinational repair have decreased viability. J Bacteriol 185:630–644 [View Article][PubMed]
     [Google Scholar]
  10. Grompe M., Versalovic J., Koeuth T., Lupski J. R. ( 1991). Mutations in the Escherichia coli dnaG gene suggest coupling between DNA replication and chromosome partitioning. J Bacteriol 173:1268–1278[PubMed]
     [Google Scholar]
  11. Guarino E., Jiménez-Sánchez A., Guzmán E. C. ( 2007a). Defective ribonucleoside diphosphate reductase impairs replication fork progression in Escherichia coli . J Bacteriol 189:3496–3501 [View Article][PubMed]
     [Google Scholar]
  12. Guarino E., Salguero I., Jiménez-Sánchez A., Guzmán E. C. ( 2007b). Double-strand break generation under deoxyribonucleotide starvation in Escherichia coli . J Bacteriol 189:5782–5786 [View Article][PubMed]
     [Google Scholar]
  13. Guzmán E. C., Caballero J. L., Jiménez-Sánchez A. ( 2002). Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli . Mol Microbiol 43:487–495 [View Article][PubMed]
     [Google Scholar]
  14. Guzmán E. C., Guarino E., Riola J., Caballero J. L., Jiménez-Sánchez A. ( 2003). Ribonucleoside diphosphate reductase is a functional and structural component of the replication hyperstructure in Escherichia coli . Recent Res Devel Mol Biol 1:29–43
     [Google Scholar]
  15. Han J. S., Kwon H. S., Yim J. B., Hwang D. S. ( 1998). Effect of IciA protein on the expression of the nrd gene encoding ribonucleoside diphosphate reductase in E. coli . Mol Gen Genet 259:610–614 [View Article][PubMed]
     [Google Scholar]
  16. Hanke P. D., Fuchs J. A. ( 1983). Regulation of ribonucleoside diphosphate reductase mRNA synthesis in Escherichia coli . J Bacteriol 154:1040–1045[PubMed]
     [Google Scholar]
  17. Herrick J., Sclavi B. ( 2007). Ribonucleotide reductase and the regulation of DNA replication: an old story and an ancient heritage. Mol Microbiol 63:22–34 [View Article][PubMed]
     [Google Scholar]
  18. Hill T. M., Sharma B., Valjavec-Gratian M., Smith J. ( 1997). sfi-independent filamentation in Escherichia coli is lexA dependent and requires DNA damage for induction. J Bacteriol 179:1931–1939[PubMed]
     [Google Scholar]
  19. Jacobson B. A., Fuchs J. A. ( 1998). Multiple cis-acting sites positively regulate Escherichia coli nrd expression. Mol Microbiol 28:1315–1322 [View Article][PubMed]
     [Google Scholar]
  20. Jaffé A., D'Ari R., Norris V. ( 1986). SOS-independent coupling between DNA replication and cell division in Escherichia coli . J Bacteriol 165:66–71[PubMed]
     [Google Scholar]
  21. Jiménez-Sánchez A., Guzmán E. C. ( 1988). Direct procedure for the determination of the number of replication forks and the reinitiation fraction in bacteria. Comput Appl Biosci 4:431–433[PubMed]
     [Google Scholar]
  22. Jordan A., Aragall E., Gibert I., Barbe J. ( 1996). Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria. Mol Microbiol 19:777–790 [View Article][PubMed]
     [Google Scholar]
  23. Kaguni J. M. ( 2006). DnaA: controlling the initiation of bacterial DNA replication and more. Annu Rev Microbiol 60:351–371 [View Article][PubMed]
     [Google Scholar]
  24. Kawakami H., Iwura T., Takata M., Sekimizu K., Hiraga S., Katayama T. ( 2001). Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the replicase and in DnaA. Mol Genet Genomics 266:167–179 [View Article][PubMed]
     [Google Scholar]
  25. Kitagawa R., Mitsuki H., Okazaki T., Ogawa T. ( 1996). A novel DnaA protein-binding site at 94.7 min on the Escherichia coli chromosome. Mol Microbiol 19:1137–1147 [View Article][PubMed]
     [Google Scholar]
  26. Kornberg A., Baker T. ( 1991). DNA replication New York: W. H. Freeman;
     [Google Scholar]
  27. Løbner-Olesen A., Slominska-Wojewodzka M., Hansen F. G., Marinus M. G. ( 2008). DnaC inactivation in Escherichia coli K-12 induces the SOS response and expression of nucleotide biosynthesis genes. PLoS ONE 3:e2984 [View Article][PubMed]
     [Google Scholar]
  28. Mathews C. K. ( 1993). Enzyme organization in DNA precursor biosynthesis. Prog Nucleic Acid Res Mol Biol 44:167–203 [View Article][PubMed]
     [Google Scholar]
  29. Messer W. ( 2002). The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication. FEMS Microbiol Rev 26:355–374[PubMed]
     [Google Scholar]
  30. Messer W., Weigel C. ( 2003). DnaA as a transcription regulator. Methods Enzymol 370:338–349 [View Article][PubMed]
     [Google Scholar]
  31. Molina F., Skarstad K. ( 2004). Replication fork and SeqA focus distributions in Escherichia coli suggest a replication hyperstructure dependent on nucleotide metabolism. Mol Microbiol 52:1597–1612 [View Article][PubMed]
     [Google Scholar]
  32. Molt K. L., Sutera V. A. Jr, Moore K. K., Lovett S. T. ( 2009). A role for nonessential domain II of initiator protein, DnaA, in replication control. Genetics 183:39–49 [View Article][PubMed]
     [Google Scholar]
  33. Monje-Casas F., Jurado J., Prieto-Alamo M. J., Holmgren A., Pueyo C. ( 2001). Expression analysis of the nrdHIEF operon from Escherichia coli. Conditions that trigger the transcript level in vivo . J Biol Chem 276:18031–18037 [View Article][PubMed]
     [Google Scholar]
  34. Morigen, Boye E., Skarstad K., Løbner-Olesen A. ( 2001). Regulation of chromosomal replication by DnaA protein availability in Escherichia coli: effects of the datA region. Biochim Biophys Acta 1521:73–80 [CrossRef]
     [Google Scholar]
  35. Morigen L.-O., Løbner-Olesen A., Skarstad K. ( 2003). Titration of the Escherichia coli DnaA protein to excess datA sites causes destabilization of replication forks, delayed replication initiation and delayed cell division. Mol Microbiol 50:349–362 [View Article][PubMed]
     [Google Scholar]
  36. Nordlund P., Reichard P. ( 2006). Ribonucleotide reductases. Annu Rev Biochem 75:681–706 [View Article][PubMed]
     [Google Scholar]
  37. Nordman J., Skovgaard O., Wright A. ( 2007). A novel class of mutations that affect DNA replication in E. coli . Mol Microbiol 64:125–138 [View Article][PubMed]
     [Google Scholar]
  38. Odsbu I., Morigen, Skarstad K. ( 2009). A reduction in ribonucleotide reductase activity slows down the chromosome replication fork but does not change its localization. PLoS ONE 4:e7617 [View Article][PubMed]
     [Google Scholar]
  39. Olliver A., Saggioro C., Herrick J., Sclavi B. ( 2010). DnaA-ATP acts as a molecular switch to control levels of ribonucleotide reductase expression in Escherichia coli . Mol Microbiol 76:1555–1571 [View Article][PubMed]
     [Google Scholar]
  40. Pato M. L. ( 1979). Alterations of deoxyribonucleoside triphosphate pools in Escherichia coli: effects on deoxyribonucleic acid replication and evidence for compartmentation. J Bacteriol 140:518–524[PubMed]
     [Google Scholar]
  41. Pritchard R. H., Zaritsky A. ( 1970). Effect of thymine concentration on the replication velocity of DNA in a thymineless mutant of Escherichia coli . Nature 226:126–131 [View Article][PubMed]
     [Google Scholar]
  42. Riola J., Guarino E., Guzmán E. C., Jiménez-Sánchez A. ( 2007). Differences in the degree of inhibition of NDP reductase by chemical inactivation and by the thermosensitive mutation nrdA101 in Escherichia coli suggest an effect on chromosome segregation. Cell Mol Biol Lett 12:70–81 [View Article][PubMed]
     [Google Scholar]
  43. Sánchez-Romero M. A., Molina F., Jiménez-Sánchez A. ( 2010). Correlation between ribonucleoside-diphosphate reductase and three replication proteins in Escherichia coli . BMC Mol Biol 11:11 [View Article][PubMed]
     [Google Scholar]
  44. Schaper S., Messer W. ( 1995). Interaction of the initiator protein DnaA of Escherichia coli with its DNA target. J Biol Chem 270:17622–17626 [View Article][PubMed]
     [Google Scholar]
  45. Skarstad K., Steen H. B., Boye E. ( 1985). Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations. J Bacteriol 163:661–668[PubMed]
     [Google Scholar]
  46. Skarstad K., von Meyenburg K., Hansen F. G., Boye E. ( 1988). Coordination of chromosome replication initiation in Escherichia coli: effects of different dnaA alleles. J Bacteriol 170:852–858[PubMed]
     [Google Scholar]
  47. Skovgaard O., Løbner-Olesen A. ( 2005). Reduced initiation frequency from oriC restores viability of a temperature-sensitive Escherichia coli replisome mutant. Microbiology 151:963–973 [View Article][PubMed]
     [Google Scholar]
  48. Stepankiw N., Kaidow A., Boye E., Bates D. ( 2009). The right half of the Escherichia coli replication origin is not essential for viability, but facilitates multi-forked replication. Mol Microbiol 74:467–479 [View Article][PubMed]
     [Google Scholar]
  49. Sueoka N., Yoshikawa H. ( 1965). The chromosome of Bacillus subtilis. I. Theory of marker frequency analysis. Genetics 52:747–757[PubMed]
     [Google Scholar]
  50. Sutera V. A. Jr, Lovett S. T. ( 2006). The role of replication initiation control in promoting survival of replication fork damage. Mol Microbiol 60:229–239 [View Article][PubMed]
     [Google Scholar]
  51. Torrents E., Grinberg I., Gorovitz-Harris B., Lundström H., Borovok I., Aharonowitz Y., Sjöberg B.-M., Cohen G. ( 2007). NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes. J Bacteriol 189:5012–5021 [View Article][PubMed]
     [Google Scholar]
  52. Tsilibaris V., Maenhaut-Michel G., Van Melderen L. ( 2006). Biological roles of the Lon ATP-dependent protease. Res Microbiol 157:701–713 [View Article][PubMed]
     [Google Scholar]
  53. Uhlin U., Eklund H. ( 1994). Structure of ribonucleotide reductase protein R1. Nature 370:533–539 [View Article][PubMed]
     [Google Scholar]
  54. Walker J. R., Severson K. A., Hermandson M. J., Blinkova A., Carr K. M., Kaguni J. M. ( 2006). Escherichia coli DnaA protein: specific biochemical defects of mutant DnaAs reduce initiation frequency to suppress a temperature-sensitive dnaX mutation. Biochimie 88:1–10 [View Article][PubMed]
     [Google Scholar]
  55. Wheeler L. J., Rajagopal I., Mathews C. K. ( 2005). Stimulation of mutagenesis by proportional deoxyribonucleoside triphosphate accumulation in Escherichia coli . DNA Repair (Amst) 4:1450–1456 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.047316-0
Loading
Overlap of replication rounds disturbs the progression of replicating forks in a ribonucleotide reductase mutant of Escherichia coli
Microbiology 157, 1955 (2011); https://doi.org/10.1099/mic.0.047316-0
/content/journal/micro/10.1099/mic.0.047316-0
/content/journal/micro/10.1099/mic.0.047316-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

PDF

Supplementary material 3

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error