Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Avoiding chromosome pathology when replication forks collide

Abstract

Chromosome duplication normally initiates through the assembly of replication fork complexes at defined origins1,2. DNA synthesis by any one fork is thought to cease when it meets another travelling in the opposite direction, at which stage the replication machinery may simply dissociate before the nascent strands are finally ligated. But what actually happens is not clear. Here we present evidence consistent with the idea that every fork collision has the potential to threaten genomic integrity. In Escherichia coli this threat is kept at bay by RecG DNA translocase3 and by single-strand DNA exonucleases. Without RecG, replication initiates where forks meet through a replisome assembly mechanism normally associated with fork repair, replication restart and recombination4,5, establishing new forks with the potential to sustain cell growth and division without an active origin. This potential is realized when roadblocks to fork progression are reduced or eliminated. It relies on the chromosome being circular, reinforcing the idea that replication initiation is triggered repeatedly by fork collision. The results reported raise the question of whether replication fork collisions have pathogenic potential for organisms that exploit several origins to replicate each chromosome.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: PriA triggers DnaA-independent chromosome replication in the absence of RecG.
Figure 2: Replisome collision triggers DnaA-independent replication.
Figure 3: Effect of RecBCD activity and oriC duplication on DnaA-independent replication.
Figure 4: Effect of recG, tus and growth phase on chromosome marker frequencies.

Accession codes

Accessions

Gene Expression Omnibus

Data deposits

Deep sequencing data have been deposited with NCBI Gene Expression Omnibus under accession number GSE41975.

References

  1. Reyes-Lamothe, R., Wang, X. & Sherratt, D. Escherichia coli and its chromosome. Trends Microbiol. 16, 238–245 (2008)

    Article  CAS  Google Scholar 

  2. Diffley, J. F. Quality control in the initiation of eukaryotic DNA replication. Phil. Trans. R. Soc. B 366, 3545–3553 (2011)

    Article  CAS  Google Scholar 

  3. Rudolph, C. J., Upton, A. L., Briggs, G. S. & Lloyd, R. G. Is RecG a general guardian of the bacterial genome? DNA Repair 9, 210–223 (2010)

    Article  CAS  Google Scholar 

  4. Gabbai, C. B. & Marians, K. J. Recruitment to stalled replication forks of the PriA DNA helicase and replisome-loading activities is essential for survival. DNA Repair 9, 202–209 (2010)

    Article  CAS  Google Scholar 

  5. Kowalczykowski, S. C. Initiation of genetic recombination and recombination-dependent replication. Trends Biochem. Sci. 25, 156–165 (2000)

    Article  CAS  Google Scholar 

  6. Kim, N. & Jinks-Robertson, S. Transcription as a source of genome instability. Nature Rev. Genet. 13, 204–214 (2012)

    Article  CAS  Google Scholar 

  7. Paul, S., Million-Weaver, S., Chattopadhyay, S., Sokurenko, E. & Merrikh, H. Accelerated gene evolution through replication-transcription conflicts. Nature 495, 512–515 (2013)

    Article  ADS  CAS  Google Scholar 

  8. Rudolph, C. J., Dhillon, P., Moore, T. & Lloyd, R. G. Avoiding and resolving conflicts between DNA replication and transcription. DNA Repair 6, 981–993 (2007)

    Article  CAS  Google Scholar 

  9. Mott, M. L. & Berger, J. M. DNA replication initiation: mechanisms and regulation in bacteria. Nature Rev. Microbiol. 5, 343–354 (2007)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Skovgaard, O., Bak, M., Lobner-Olesen, A. & Tommerup, N. Genome-wide detection of chromosomal rearrangements, indels, and mutations in circular chromosomes by short read sequencing. Genome Res. 21, 1388–1393 (2011)

    Article  CAS  Google Scholar 

  12. Trautinger, B. W., Jaktaji, R. P., Rusakova, E. & Lloyd, R. G. RNA polymerase modulators and DNA repair activities resolve conflicts between DNA replication and transcription. Mol. Cell 19, 247–258 (2005)

    Article  CAS  Google Scholar 

  13. Dutta, D., Shatalin, K., Epshtein, V., Gottesman, M. E. & Nudler, E. Linking RNA polymerase backtracking to genome instability in E. coli. Cell 146, 533–543 (2011)

    Article  CAS  Google Scholar 

  14. Gregg, A. V., McGlynn, P., Jaktaji, R. P. & Lloyd, R. G. Direct rescue of stalled DNA replication forks via the combined action of PriA and RecG helicase activities. Mol. Cell 9, 241–251 (2002)

    Article  CAS  Google Scholar 

  15. Rudolph, C. J., Mahdi, A. A., Upton, A. L. & Lloyd, R. G. RecG protein and single-strand DNA exonucleases avoid cell lethality associated with PriA helicase activity in Escherichia coli. Genetics 186, 473–492 (2010)

    Article  CAS  Google Scholar 

  16. Cui, T. et al. Escherichia coli with a linear genome. EMBO Rep. 8, 181–187 (2007)

    Article  ADS  CAS  Google Scholar 

  17. Wang, X., Lesterlin, C., Reyes-Lamothe, R., Ball, G. & Sherratt, D. J. Replication and segregation of an Escherichia coli chromosome with two replication origins. Proc. Natl Acad. Sci. USA 108, E243–E250 (2011)

    Article  ADS  Google Scholar 

  18. Mahdi, A. A., Briggs, G. S. & Lloyd, R. G. Modulation of DNA damage tolerance in Escherichia coli recG and ruv strains by mutations affecting PriB, the ribosome and RNA polymerase. Mol. Microbiol. 86, 675–691 (2012)

    Article  CAS  Google Scholar 

  19. Fachinetti, D. et al. Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements. Mol. Cell 39, 595–605 (2010)

    Article  CAS  Google Scholar 

  20. Steinacher, R., Osman, F., Dalgaard, J. Z., Lorenz, A. & Whitby, M. C. The DNA helicase Pfh1 promotes fork merging at replication termination sites to ensure genome stability. Genes Dev. 26, 594–602 (2012)

    Article  CAS  Google Scholar 

  21. Ralf, C., Hickson, I. D. & Wu, L. The Bloom’s syndrome helicase can promote the regression of a model replication fork. J. Biol. Chem. 281, 22839–22846 (2006)

    Article  CAS  Google Scholar 

  22. Whitby, M. C. The FANCM family of DNA helicases/translocases. DNA Repair 9, 224–236 (2010)

    Article  CAS  Google Scholar 

  23. Betous, R. et al. SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication. Genes Dev. 26, 151–162 (2012)

    Article  CAS  Google Scholar 

  24. Killen, M. W., Stults, D. M., Wilson, W. A. & Pierce, A. J. Escherichia coli RecG functionally suppresses human Bloom syndrome phenotypes. BMC Mol. Biol. 13, 33 (2012)

    Article  CAS  Google Scholar 

  25. Kornblum, C. et al. Loss-of-function mutations in MGME1 impair mtDNA replication and cause multisystemic mitochondrial disease. Nature Genet. 45, 214–219 (2013)

    Article  CAS  Google Scholar 

  26. Müller, C. A. & Nieduszynski, C. A. Conservation of replication timing reveals global and local regulation of replication origin activity. Genome Res. 22, 1953–1962 (2012)

    Article  Google Scholar 

  27. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000)

    ADS  CAS  Google Scholar 

  28. McGlynn, P. & Lloyd, R. G. Modulation of RNA polymerase by (p)ppGpp reveals a RecG-dependent mechanism for replication fork progression. Cell 101, 35–45 (2000)

    Article  CAS  Google Scholar 

  29. Miller, J. H. Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, 1972)

    Google Scholar 

  30. Rudolph, C. J., Upton, A. L. & Lloyd, R. G. Replication fork collisions cause pathological chromosomal amplification in cells lacking RecG DNA translocase. Mol. Microbiol. 74, 940–955 (2009)

    Article  CAS  Google Scholar 

  31. Rybchin, V. N. & Svarchevsky, A. N. The plasmid prophage N15: a linear DNA with covalently closed ends. Mol. Microbiol. 33, 895–903 (1999)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank T. Horiuchi and D. Sherratt for E. coli strains, A. Mahdi for help with DNA extractions, S. Malla and M. Blythe for deep sequencing, S. Demolli and D. Ivanova for control experiments, and C. Buckman and L. Harris for assistance. This work was supported by grants from the MRC (R.G.L., G0800970), the Leverhulme Trust (C.J.R.) and the BBSRC (C.A.N., BB/E023754/1).

Author information

Authors and Affiliations

Authors

Contributions

C.J.R. and R.G.L. initiated and directed the project. C.J.R., A.L.U., A.S., C.A.N. and R.G.L. performed the experimental work. C.J.R., A.L.U., C.A.N. and R.G.L. analysed the data and wrote the paper.

Corresponding author

Correspondence to Christian J. Rudolph.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1, Supplementary Figures 1-8, a Supplementary Discussion and Supplementary References. (PDF 4884 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Rudolph, C., Upton, A., Stockum, A. et al. Avoiding chromosome pathology when replication forks collide. Nature 500, 608–611 (2013). https://doi.org/10.1038/nature12312

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12312

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing