Skip to main content
SpringerLink
Log in

Identification of temperature-sensitive dnaD mutants of Staphylococcus aureus that are defective in chromosomal DNA replication

  • Original Paper
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

The DnaD protein in Gram-positive bacteria is thought to be essential for the initiation step in DNA replication. In the present study, we characterized two Staphylococcus aureus mutants whose temperature-sensitive growth phenotype could be complemented by a plasmid carrying the dnaD gene. These mutants each had a single amino acid substitution in the DnaD protein and showed decreased DNA synthesis at restrictive temperature. Analyses of the origin to terminus ratio by Southern blotting, and of origin numbers per cell by flow cytometry, revealed that, at the restrictive temperature, one mutant continued ongoing DNA replication but failed to initiate DNA replication. The other mutant, in contrast, could not complete ongoing DNA replication and proceeded to degrade the chromosome. However, if protein synthesis was inhibited, the second mutant could complete DNA replication. These results suggest that DnaD protein is necessary not only for the initiation step, but also to avoid replication fork blockage. Moreover, both mutants were sensitive to mitomycin C, a drug that induces DNA damage, suggesting that the DnaD protein is also involved in DNA repair.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5A, B

Similar content being viewed by others

References

  • Allen GC Jr, Kornberg A (1991) Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli. J Biol Chem 266:22096–22101

    CAS  PubMed  Google Scholar 

  • Bird LE, Pan H, Soultanas P, Wigley DB (2000) Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus. Biochemistry 39:171–182

    Article  CAS  PubMed  Google Scholar 

  • Bouche JP, Zechel K, Kornberg A (1975) dnaG gene product, a rifampicin-resistant RNA polymerase, initiates the conversion of a single-stranded coliphage DNA to its duplex replicative form. J Biol Chem 250:5995–6001

    CAS  PubMed  Google Scholar 

  • Bramhill D, Kornberg A (1988) Duplex opening by DnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell 52:743–755

    CAS  Google Scholar 

  • Bruand C, Ehrlich SD (1995) The Bacillus subtilis dnaI gene is part of the dnaB operon. Microbiology 141:1199–1200

    CAS  PubMed  Google Scholar 

  • Bruand C, Ehrlich SD, Janniere L (1995a) Primosome assembly site in Bacillus subtilis. EMBO J 14:2642–2650

    CAS  PubMed  Google Scholar 

  • Bruand C, Sorokin A, Serror P, Ehrlich SD (1995b) Nucleotide sequence of the Bacillus subtilis dnaD gene. Microbiology 141:321–322

    CAS  PubMed  Google Scholar 

  • Bruand C, Bidnenko V, Ehrlich SD (2001a) Replication mutations differentially enhance RecA-dependent and RecA-independent recombination between tandem repeats in Bacillus subtilis. Mol Microbiol 39:1248–1258

    Article  CAS  PubMed  Google Scholar 

  • Bruand C, Farache M, McGovern S, Ehrlich SD, Polard P (2001b) DnaB, DnaD and DnaI proteins are components of the Bacillus subtilis replication restart primosome. Mol Microbiol 42:245–255

    Article  CAS  PubMed  Google Scholar 

  • Carl PL (1970) Escherichia coli mutants with temperature-sensitive synthesis of DNA. Mol Gen Genet 109:107–122

    CAS  PubMed  Google Scholar 

  • Cox MM, Goodman MF, Kreuzer KN, Sherratt DJ, Sandler SJ, Marians KJ (2000) The importance of repairing stalled replication forks. Nature 404:37–41

    CAS  PubMed  Google Scholar 

  • Ehrlich SD (1977) Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc Natl Acad Sci USA 74:1680–1682

    CAS  PubMed  Google Scholar 

  • Fujimura T, Murakami K (1997) Increase of methicillin resistance in Staphylococcus aureus caused by deletion of a gene whose product is homologous to lytic enzymes. J Bacteriol 179:6294–6301

    CAS  PubMed  Google Scholar 

  • Hiasa H, Marians KJ (1999) Initiation of bidirectional replication at the chromosomal origin is directed by the interaction between helicase and primase. J Biol Chem 274:27244–27248

    Article  CAS  PubMed  Google Scholar 

  • Ichihashi N, Kurokawa K, Matsuo M, Kaito C, Sekimizu K (2003) Inhibitory effects of basic or neutral phospholipid on acidic phospholipid-mediated dissociation of adenine nucleotide bound to DnaA protein, the initiator of chromosomal DNA replication. J Biol Chem 278:28778–28786

    Article  CAS  PubMed  Google Scholar 

  • Imai Y, Ogasawara N, Ishigo-Oka D, Kadoya R, Daito T, Moriya S (2000) Subcellular localization of DNA-initiation proteins of Bacillus subtilis: evidence that chromosome replication begins at either edge of the nucleoids. Mol Microbiol 36:1037–1048

    Article  CAS  PubMed  Google Scholar 

  • Inoue R, Kaito C, Tanabe M, Kamura K, Akimitsu N, Sekimizu K (2001) Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus. Mol Genet Genomics 266:564–571

    Article  CAS  PubMed  Google Scholar 

  • Ishigo-Oka D, Ogasawara N, Moriya S (2001) DnaD protein of Bacillus subtilis interacts with DnaA, the initiator protein of replication. J Bacteriol 183:2148–2150

    Article  CAS  PubMed  Google Scholar 

  • Kaito C, Kurokawa K, Hossain MS, Akimitsu N, Sekimizu K (2002) Isolation and characterization of temperature-sensitive mutants of the Staphylococcus aureus dnaC gene. FEMS Microbiol Lett 210:157–164

    Article  CAS  PubMed  Google Scholar 

  • Karamata D, Gross JD (1970) Isolation and genetic analysis of temperature-sensitive mutants of B. subtilis defective in DNA synthesis. Mol Gen Genet 108:277–287

    CAS  PubMed  Google Scholar 

  • Kelman Z, O’Donnell M (1995) DNA polymerase III holoenzyme: structure and function of a chromosomal replicating machine. Annu Rev Biochem 64:171–200

    Article  CAS  PubMed  Google Scholar 

  • Kogoma T (1997) Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription. Microbiol Mol Biol Rev 61:212–238

    CAS  PubMed  Google Scholar 

  • Kogoma T, Cadwell GW, Barnard KG, Asai T (1996) The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair. J Bacteriol 178:1258–1264

    CAS  PubMed  Google Scholar 

  • Kornberg A, Baker T (1992) DNA Replication. WH Freeman, New York

  • Kowalczykowski SC (2000) Initiation of genetic recombination and recombination-dependent replication. Trends Biochem Sci 25:156–165

    Article  CAS  PubMed  Google Scholar 

  • Kunst F, et al (1997) The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390:249–256

    CAS  PubMed  Google Scholar 

  • Kuroda M, et al (2001) Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357:1225–1240

    Article  CAS  PubMed  Google Scholar 

  • Lark CA, Riazi J, Lark KG (1978) dnaT, a dominant conditional-lethal mutation affecting DNA replication in Escherichia coli. J Bacteriol 136:1008–1017

    CAS  PubMed  Google Scholar 

  • Lemon KP, Grossman AD (1998) Localization of bacterial DNA polymerase: evidence for a factory model of replication. Science 282:1516–1519

    Article  CAS  PubMed  Google Scholar 

  • Lu YB, Ratnakar PV, Mohanty BK, Bastia D (1996) Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA. Proc Natl Acad Sci USA 93:12902–12907

    Article  CAS  PubMed  Google Scholar 

  • Marians KJ (2000) PriA-directed replication fork restart in Escherichia coli. Trends Biochem Sci 25:185–189

    Article  CAS  PubMed  Google Scholar 

  • Marsin S, McGovern S, Ehrlich SD, Bruand C, Polard P (2001) Early steps of Bacillus subtilis primosome assembly. J Biol Chem 276:45818–45825

    Article  CAS  PubMed  Google Scholar 

  • Moriya S, Imai Y, Hassan AK, Ogasawara N (1999) Regulation of initiation of Bacillus subtilis chromosome replication. Plasmid 41:17–29

    Article  CAS  PubMed  Google Scholar 

  • Ng JY, Marians KJ (1996) The ordered assembly of the phiX174-type primosome. I. Isolation and identification of intermediate protein-DNA complexes. J Biol Chem 271:15642–15648

    Article  CAS  PubMed  Google Scholar 

  • Ogasawara N, Moriya S, Mazza PG, Yoshikawa H (1986) Nucleotide sequence and organization of dnaB gene and neighbouring genes on the Bacillus subtilis chromosome. Nucleic Acids Res 14:9989–9999

    CAS  PubMed  Google Scholar 

  • Polard P, Marsin S, McGovern S, Velten M, Wigley DB, Ehrlich SD, Bruand C (2002) Restart of DNA replication in Gram-positive bacteria: functional characterisation of the Bacillus subtilis PriA initiator. Nucleic Acids Res 30:1593–1605

    Article  CAS  PubMed  Google Scholar 

  • Schekman R, Weiner JH, Weiner A, Kornberg A (1975) Ten proteins required for conversion of phiX174 single-stranded DNA to duplex form in vitro. Resolution and reconstitution. J Biol Chem 250:5859–5865

    CAS  PubMed  Google Scholar 

  • Sekimizu K, Bramhill D, Kornberg A (1988) Sequential early stages in the in vitro initiation of replication at the origin of the Escherichia coli chromosome. J Biol Chem 263:7124–7130

    CAS  PubMed  Google Scholar 

  • Skarstad K, Wold S (1995) The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. Mol Microbiol 17:825–831

    Article  CAS  PubMed  Google Scholar 

  • Skarstad K, Bernander R, Boye E (1995) Analysis of DNA replication in vivo by flow cytometry. Methods Enzymol 262:604–613

    CAS  PubMed  Google Scholar 

  • Soultanas P (2002) A functional interaction between the putative primosomal protein DnaI and the main replicative DNA helicase DnaB in Bacillus. Nucleic Acids Res 30:966–974

    Article  CAS  PubMed  Google Scholar 

  • Tougu K, Peng H, Marians KJ (1994) Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork. J Biol Chem 269:4675–4682

    CAS  PubMed  Google Scholar 

  • Velten M, McGovern S, Marsin S, Ehrlich SD, Noirot P, Polard P (2003) A two-protein strategy for the functional loading of a cellular replicative DNA helicase. Mol Cell 11:1009–1020

    CAS  PubMed  Google Scholar 

  • Wechsler JA (1975) Genetic and phenotypic characterization of dnaC mutations. J Bacteriol 121:594–599

    CAS  PubMed  Google Scholar 

  • Wechsler JA, Gross JD (1971) Escherichia coli mutants temperature-sensitive for DNA synthesis. Mol Gen Genet 113:273–284

    CAS  PubMed  Google Scholar 

  • Wickner S (1977) DNA or RNA priming of bacteriophage G4 DNA synthesis by Escherichia coli dnaG protein. Proc Natl Acad Sci U S A 74:2815–2819

    CAS  PubMed  Google Scholar 

  • Wickner S, Hurwitz J (1974) Conversion of phiX174 viral DNA to double-stranded form by purified Escherichia coli proteins. Proc Natl Acad Sci USA 71:4120–4124

    CAS  PubMed  Google Scholar 

  • Wu CA, Zechner EL, Marians KJ (1992a) Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size. J Biol Chem 267:4030–4044

    CAS  PubMed  Google Scholar 

  • Wu CA, Zechner EL, Reems JA, McHenry CS, Marians KJ (1992b) Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. V. Primase action regulates the cycle of Okazaki fragment synthesis. J Biol Chem 267:4074–4083

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research of Japan Society for the Promotion of Science (JSPS). We thank Misses Yoriko Okada, Teruko Aota, Makiko Miyatani, Hiromi Komaki and Kozue Saito for their technical assistance

Author information

Author notes

  1. N. Fukuhara

    Present address: Shionogi Laboratory of Biomolecular Chemistry, Frontier Research Center for Post-Genomic Science and Technology, Graduate School of Science, Hokkaido University, Kita-21, Nishi-11, 001-0021, Sapporo, Japan

Authors and Affiliations

  1. Laboratory of Developmental Biochemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 3-1, 7-Chome, Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan

    Y. Li, K. Kurokawa, M. Matsuo & K. Sekimizu

  2. Discovery Research Laboratories, Shionogi and Co. Ltd., 3-1-1, Futaba-Cho, 561-0825, Toyonaka, Japan

    N. Fukuhara & K. Murakami

Authors
  1. Y. Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. K. Kurokawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Matsuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. N. Fukuhara
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K. Murakami
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. K. Sekimizu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to K. Kurokawa.

Additional information

Communicated by H. Ikeda

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, Y., Kurokawa, K., Matsuo, M. et al. Identification of temperature-sensitive dnaD mutants of Staphylococcus aureus that are defective in chromosomal DNA replication. Mol Genet Genomics 271, 447–457 (2004). https://doi.org/10.1007/s00438-004-0996-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-004-0996-6

Keywords

Search

Navigation