Abstract
Single-molecule analyses of DNA replication have greatly advanced our understanding of mammalian replication restart. Several proteins that are not part of the core replication machinery promote the efficient restart of replication forks that have been stalled by replication inhibitors, suggesting that bona fide fork restart pathways exist in mammalian cells. Different models of replication fork restart can be envisaged, based on the involvement of DNA helicases, nucleases, homologous recombination factors and the importance of DNA double-strand break formation.
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Acknowledgements
We thank the Medical Research Council, the Swedish Research Council, the Swedish Children's Cancer Foundation, the Swedish Pain Relief Foundation and the Swedish Cancer Society for financial support.
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Supplementary information
Supplementary information 1 (box)
Replication fork restart in E. coli (PDF 76 kb)
Supplementary information 2 (box)
Chromosome combing and DNA fiber technique (PDF 59 kb)
Supplementary information 3 (box)
Replication fork stabilisation (PDF 80 kb)
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Glossary
- Aphidicolin
-
A small-molecule inhibitor that directly blocks the activity of the replicative DNA polymerases. Aphidicolin treatment leads to replication fork stalling and eventually DNA DSB formation.
- Break-induced replication
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A mechanism for origin-independent replication restart, whereby a resected DNA end invades a homologous DNA molecule, thus establishing a replication fork.
- Chromosome combing
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A method for single-molecule analysis of DNA replication forks. Newly replicated DNA is labelled in vivo using halogenated thymidine analogues and genomic DNA is isolated and spread out on microscope coverslips. Immunofluorescence staining of the thymidine analogues is used to visualize the labelled tracks that are left on the DNA by moving replication forks.
- Core replication machinery
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The complex of proteins that is essential for all DNA replication and includes the replicative DNA helicase, primase, clamp loader, sliding clamp and leading- and lagging-strand DNA polymerases.
- DNA fibre technique
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A technique that is similar to chromosome combing, but in which cells on microscope slides are treated with detergent and the DNA is spread directly out of the lysed nuclei. Because of the lysis method, this technique is used in vertebrate cells but not in yeast.
- DNA helicase
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An enzyme that translocates on DNA and unwinds the double helix into ssDNA in an ATP-driven reaction. Annealing helicases use ATP to catalyse the reverse reaction.
- Holliday junction
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A four-way junction between two dsDNA molecules of homologous sequence. Holliday junctions are mobile and can be translocated by DNA helicases (branch migration).
- Hydroxyurea
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A radical scavenger that inhibits ribonucleotide reductase, which results in cells producing less of the desoxyribonucleotides that are used for DNA synthesis. Hydroxyurea treatment leads to replication fork stalling and eventually DNA DSB formation.
- Lagging strand
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The nascent strand of DNA that is synthesized discontinuously in short pieces (Okazaki fragments) at the replication fork.
- Origin firing
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The start of replication fork progression at a replication origin. Mammalian origin firing is restricted to S phase and is controlled by cell cycle signalling.
- Replication fork
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A structure formed when the template strands have been separated by helicases and a newly formed copy of the DNA is synthesized. The fork moves in the direction of leading-strand synthesis.
- Replication fork restart
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The resumption of fork progression after removal or bypass of a replication block.
- Replication fork stabilization
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The maintenance of viable replication fork structures and the replication machinery during a replication block.
- Replication origin
-
The chromosomal location at which new replication factories are assembled and replication is initiated. Replication fork movement from origins is bidirectional.
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Petermann, E., Helleday, T. Pathways of mammalian replication fork restart. Nat Rev Mol Cell Biol 11, 683–687 (2010). https://doi.org/10.1038/nrm2974
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DOI: https://doi.org/10.1038/nrm2974
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