Skip to main content
SpringerLink
Log in

Localization of replication forks in wild-type and mukB mutant cells of Escherichia coli

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

Abstract

To examine the subcellular localization of the replication machinery in Escherichia coli, we have developed an immunofluorescence method that allows us to determine the subcellular location of newly synthesized DNA pulse-labeled with 5-bromo-2′-deoxyuridine (BrdU). Using this technique, we have analyzed growing cells. In wild-type cells that showed a single BrdU fluorescence signal, the focus was located in the middle of the cell; in cells with two signals, the foci were localized at positions equivalent to 1/4 and 3/4 of the cell length. The formation of BrdU foci was dependent upon ongoing chromosomal replication. A mutant lacking MukB, which is required for proper partitioning of sister chromosomes, failed to maintain the ordered localization of BrdU foci: (1) a single BrdU focus tended to be localized at a pole-proximal region of the nucleoid, and (2) a focus was often found to consist of two replicating chromosomes. Thus, the positioning of replication forks is affected by the disruption of the mukB gene.

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

Similar content being viewed by others

References

  • Adachi S, Hiraga S (2003) Mutants suppressing novobiocin hypersensitivity of a mukB null mutant. J Bacteriol 185:3690–3695

    Article  PubMed  CAS  Google Scholar 

  • Blaauwen TD, Lindqvist A, Löwe J, Nanninga N (2001) Distribution of the Escherichia coli structural maintenance of chromosomes (SMC)-like protein MukB in the cell. Mol Microbiol 42:1179–1188

    Article  PubMed  Google Scholar 

  • Brendler T, Austin S (1999) Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences. EMBO J 18:2304–2310

    Article  PubMed  CAS  Google Scholar 

  • Brendler T, Sawitzke J, Sergueev K, Austin S (2000) A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation. EMBO J 19:6249–6258

    Article  PubMed  CAS  Google Scholar 

  • Britton RA, Lin CD, Grossman AD (1998) Characterization of a prokaryotic SMC protein involved in chromosome partitioning. Genes Dev 12:1254–1259

    PubMed  CAS  Google Scholar 

  • Gordon GS, Sitnikov D, Webb CD, teleman A, Straight A, Losick R, Murray AW, Wright A (1997) Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechanisms. Cell 90:1113–1121

    Article  PubMed  CAS  Google Scholar 

  • Graumann PL, Losick R, Strunnikov AV (1998) Subcellular localization of Bacillus subtilis SMC, a protein involved in chromosome condensation and segregation. J Bacteriol 180:5749–5755

    PubMed  CAS  Google Scholar 

  • Hiraga S (2000) Dynamic localization of bacterial and plasmid chromosomes. Annu Rev Genet 34:21–59

    Article  PubMed  CAS  Google Scholar 

  • Hiraga S, Niki H, Ogura T, Ichinose C, Mori H, Ezaki B, Jaffé A (1989) Chromosome partitioning in Escherichia coli: novel mutants producing anucleate cells. J Bacteriol 171:1496–1505

    PubMed  CAS  Google Scholar 

  • Hiraga S, Ichinose C, Niki H, Yamazoe M (1998) Cell cycle-dependent duplication and bidirectional migration of SeqA-associated DNA-protein complexes in E. coli. Mol Cell 1:381–387

    Article  PubMed  CAS  Google Scholar 

  • Hiraga S, Ichinose C, Onogi T, Niki H, Yamazoe M (2000) Bidirectional migration of SeqA-bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli. Genes Cells 5:327–341

    Article  PubMed  CAS  Google Scholar 

  • Hirano T (1999) SMC-mediated chromosome mechanisms: a conserved scheme from bacteria to vertebrates? Genes Dev 13:11–19

    PubMed  CAS  Google Scholar 

  • Hirano T, Mitchison TJ, Swedlow JR (1995) The SMC family: from chromosome condensation to dosage compensation. Curr Opin Cell Biol 7:329–336

    Article  PubMed  CAS  Google Scholar 

  • Holmes VF, Cozzarelli NR (2000) Closing the ring: links between SMC proteins and chromosome partitioning, condensation, and supercoiling. Proc Natl Acad Sci USA 97:1322–1324

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Lewis PJ, Errington J (1997) Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and co-localization with the Spo0J partitioning protein. Mol Microbiol 25:945–954

    Article  PubMed  CAS  Google Scholar 

  • Lindow JC, Britton RA, Grossman AD (2002a) Structural maintenance of chromosomes protein of Bacillus subtilis affects supercoiling in vivo. J Bacteriol 184:5317–5322

    Article  PubMed  CAS  Google Scholar 

  • Lindow JC, Kuwano M, Moriya S, Grossman AD (2002b) Subcellular localization of the Bacillus subtilis structural maintenance of chromosomes (SMC) protein. Mol Microbiol 46:997–1007

    Article  PubMed  CAS  Google Scholar 

  • Lu M, Campbel JL, Boye E, Kleckner N (1994) SeqA: a negative modulator of replication initiation in E. coli. Cell 77:413–426

    Article  PubMed  CAS  Google Scholar 

  • Mascarenhas J, Soppa J, Strunnikov AV, Graumann PL (2002) Cell cycle-dependent localization of two novel prokaryotic chromosome segregation and condensation proteins in Bacillus subtilis that interact with SMC protein. EMBO J 21:3108–3118

    Article  PubMed  CAS  Google Scholar 

  • Melby TE, Ciampaglio CN, Briscore B, Erickson HP (1998) The symmetrical structure of structure maintenance of chromosomes (SMC) and MukB proteins: long, antiparallel coiled coils, folded at a flexible hinge. J Cell Biol 142:1595–1604

    Article  PubMed  CAS  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Moriya S, Tsujikawa E, Hassan AK, Asai K, Kodama T, Ogasawara N (1998) A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition. Mol Microbiol 29:179–187

    Article  PubMed  CAS  Google Scholar 

  • Niki H, Hiraga S (1998) Polar localization of the replication origin and terminus in Escherichia coli nucleoids during chromosome partitioning. Genes Dev 12:1036–1045

    PubMed  CAS  Google Scholar 

  • Niki H, Jaffé A, Imamura R, Ogura T, Hiraga S (1991) The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli. EMBO J 10:183–193

    PubMed  CAS  Google Scholar 

  • Niki H, Imamura R, Kitaoka M, Yamanaka K, Ogura T, Hiraga S (1992) E. coli MukB protein involved in chromosome partition forms a homodimer with a rod-and hinge structure having DNA binding and ATP/GTP binding activities. EMBO J 11:5101–5109

    PubMed  CAS  Google Scholar 

  • Niki H, Yamaichi Y, Hiraga S (2000) Dynamic organization of chromosomal DNA in Escherichia coli. Genes Dev 14:212–223

    PubMed  CAS  Google Scholar 

  • Ohsumi K, Yamazoe M, Hiraga S (2001) Different localization of SeqA-bound nascent DNA clusters and MukF-MukE-MukB complex in Escherichia coli cells. Mol Microbiol 40:835–845

    Article  PubMed  CAS  Google Scholar 

  • Onogi T, Niki H, Yamazoe M, Hiraga S (1999) The assembly and migration of SeqA-GFP fusion in living cells of Escherichia coli. Mol Microbiol 31:1775–1782

    Article  PubMed  CAS  Google Scholar 

  • Onogi T, Yamazoe M, Ichinose C, Niki H, Hiraga S (2000) Null mutation of the dam or seqA gene suppresses temperature-sensitive lethality but not hypersensitivity to novobiocin of muk null mutants. J Bacteriol 182:5898–5901

    Article  PubMed  CAS  Google Scholar 

  • Onogi T, Ohsumi K, Katayama T, Hiraga S (2002) Replication-dependent recruitment of the β-subunit of DNA polymerase III from cytosolic spaces to replication forks in Escherichia coli. J Bacteriol 184:867–870

    Article  PubMed  CAS  Google Scholar 

  • Sawitzke JA, Austin S (2000) Suppression of chromosome segregation defects of Escherichia coli muk mutants by mutations in topoisomerase I. Proc Natl Acad Sci USA 97:1671–1676

    Article  PubMed  CAS  Google Scholar 

  • Skarstad K, Steen BH, Boye E (1985) Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations. J Bacteriol 163:661–668

    PubMed  CAS  Google Scholar 

  • Slater S, Wold S, Lu M, Boye E, Skarstad K, Kleckner N (1995) E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration. Cell 82:927–936

    Article  PubMed  CAS  Google Scholar 

  • Soppa J, Derryn E, Ogasawara N, Moriya S (2002) Discovery of two novel families of proteins that are proposed to interact with prokaryotic SMC proteins, and characterization of the Bacillus subtilis family members ScpA and ScpB. Mol Microbiol 45:59–71

    Article  PubMed  CAS  Google Scholar 

  • Sunako Y, Onogi T, Hiraga S (2001) Sister chromosome cohesion of Escherichia coli. Mol Microbiol 42:1233–1241

    Article  PubMed  CAS  Google Scholar 

  • Weitao T, Nordström K, Dasgupta S (1999) Mutual suppression of mukB and seqA phenotypes might arise from their opposing influences on the Escherichia coli nucleoid structure. Mol Microbiol 34:157–168

    Article  PubMed  CAS  Google Scholar 

  • Weitao T, Dasgupta S, Nordström K (2000a) Role of the mukB gene in chromosome and plasmid partition in Escherichia coli. Mol Microbiol 38:392–400

    Article  PubMed  CAS  Google Scholar 

  • Weitao T, Nordström K, Dasgupta S (2000b) Escherichia coli cell cycle control genes affect chromosome superhelicity. EMBO Rep 1:494–499

    PubMed  CAS  Google Scholar 

  • Yamanaka K, Ogura T, Niki H, Hiraga S (1996) Identification of two new genes, mukE and mukF, involved in chromosome partitioning in Escherichia coli. Mol Gen Genet 250:241–251

    PubMed  CAS  Google Scholar 

  • Yamazoe M, Onogi T, Sunako Y, Niki H, Yamanaka K, Ichimura T, Hiraga S (1999) Complex formation of MukB, MukE and MukF proteins involved in chromosome partitioning in Escherichia coli. EMBO J 18:5873–5884

    Article  PubMed  CAS  Google Scholar 

  • Yamazoe M, Adachi S, Kanaya S, Ohsumi K, Hiraga S (2005) Sequential binding of SeqA protein to nascent DNA segments at replication forks in synchronized cultures of Escherichia coli. Mol Microbiol 55:289–298

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

M. K. is indebted to M. C. Gendron for flow cytometry, to J. Meury, G. Geaud, and C. Chamot for fluorescence microscopy, to the Association pour la Recherche Scientifique contre les Cancers for financial support, and to D. Ekiert for careful reading of the manuscript. S. H. is supported by a grant from the Center of Excellence (COE) of Kyoto University, and by grants from the Ministry of Education, Science, Sports, Culture and Technology of Japan. S. A. is supported by a fellowship and grant from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

Author information

Authors and Affiliations

  1. Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyo-ku, Kyoto, 606-8501, Japan

    Shun Adachi & Sota Hiraga

  2. Institut Jacques Monod, Université Paris 6-7, CNRS, 2 Place Jussieu, 75251, Paris Cedex 05, France

    Masamichi Kohiyama

  3. Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 4-24-1 Kuhonji, Kumamoto, 862-0976, Japan

    Toshinari Onogi & Sota Hiraga

  4. Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuou-ku, Chiba, 260-8673, Japan

    Toshinari Onogi

Authors
  1. Shun Adachi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Masamichi Kohiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Toshinari Onogi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sota Hiraga
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sota Hiraga.

Additional information

Communicated by W. Goebel

Rights and permissions

Reprints and permissions

About this article

Cite this article

Adachi, S., Kohiyama, M., Onogi, T. et al. Localization of replication forks in wild-type and mukB mutant cells of Escherichia coli . Mol Genet Genomics 274, 264–271 (2005). https://doi.org/10.1007/s00438-005-0023-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-005-0023-6

Keywords

Search

Navigation