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  • Review Article
  • Published:

Preventing re-replication of chromosomal DNA

Key Points

  • To ensure precise duplication of chromosomal DNA, replication origins are 'licensed' for replication during late mitosis and early G1 by loading complexes of the Mcm2–7 proteins. The licensing system is turned off during S phase and G2, thereby ensuring that no replication origins can fire more than once in a single cell cycle.

  • Recent structural studies have revealed that the Mcm2–7 proteins form a hexameric ring that can potentially encircle double-stranded DNA. The ability of Mcm2–7 to be clamped around DNA potentially explains why the licensed state can be stably maintained over long time periods. The Mcm2–7 structure resembles that of SV40 T antigen, which is a replicative helicase, consistent with the idea that Mcm2–7 functions as a replicative helicase.

  • Recent evidence indicates that the licensing system is regulated in different ways in different organisms. In yeasts, high CDK activity from late G1 until the end of mitosis inhibits different components of the licensing system in a range of different ways. In metazoans, however, the Cdt1 component of the licensing system seems to be the main component that is down-regulated late in the cell cycle. On progression into S phase, metazoan Cdt1 is degraded, and a Cdt1 inhibitor called geminin becomes active. If metazoans over-express Cdt1 in S phase, or lack geminin, re-replication of DNA occurs.

  • In cells that are unable to down-regulate the licensing system in S phase and G2, re-replication of DNA takes place. Metazoan cells respond to this by activating a number of different checkpoint pathways, which cause cell-cycle arrest or apoptosis.

  • When cells exit from the cell cycle, Mcm2–7 and other components of the licensing system are degraded. This potentially provides a barrier to prevent the inappropriate proliferation of such cells. To re-enter the cell-cycle, quiescent cells must first re-license their DNA. Recent work has shown that mouse embryos lacking cyclins E1 and E2 are unable to re-license their DNA on exit from quiescence. The cyclin E-null embryos are also unable to produce some cell types that are normally polyploid. These results indicate that cyclin E has a central role in activating the licensing system.

Abstract

To ensure its duplication, chromosomal DNA must be precisely duplicated in each cell cycle, with no sections left unreplicated, and no sections replicated more than once. Eukaryotic cells achieve this by dividing replication into two non-overlapping phases. During late mitosis and G1, replication origins are 'licensed' for replication by loading the minichromosome maintenance (Mcm) 2–7 proteins to form a pre-replicative complex. Mcm2–7 proteins are then essential for initiating and elongating replication forks during S phase. Recent data have provided biochemical and structural insight into the process of replication licensing and the mechanisms that regulate it during the cell cycle.

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Figure 1: Regulated loading and unloading of Mcm2–7 during the cell cycle.
Figure 2: Crystal structure of the N terminus of the Methanobacter thermoautotrophicum MCM.
Figure 3: Model for SV40 T antigen function at replication initiation.
Figure 4: Stepwise assembly of pre-replicative complex proteins during origin licensing.
Figure 5: Cell-cycle regulation of the licensing system in yeasts and metazoans.
Figure 6: Structure of a geminin–Cdt1 complex.
Figure 7: The possible role of cyclin E in promoting re-licensing of DNA on exit from G0.

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Acknowledgements

Thanks to Margret Michalski-Blow for assembling the glossary. This work was supported by a Cancer Research UK grant and National Institutes of Health grants.

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Correspondence to J. Julian Blow.

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DATABASES

Swiss-Prot

ATM kinase

ATR kinase

Cdc6

Cdc6

CDK2

CDK4

Cdt1

Chk1

Chk2

cyclin A

cyclin E

Geminin

Mcm2

Mcm3

Mcm4

Mcm5

Mcm6

Mcm7

FURTHER INFORMATION

School of Life Sciences Research at the University of Dundee

Dutta laboratory

Blow laboratory

Glossary

DNA HELICASE

A class of enzyme that uses NTP hydrolysis to transiently separate double-stranded DNA into two single DNA strands.

ATPase MOTIF

A group of conserved amino-acid sequences that are predicted to form a structure that can bind and hydrolyze ATP.

TOPOISOMERASE

A class of enzymes that can alter the supercoiling of double-stranded DNA by transiently cutting one or both strands of the DNA.

INTRA-S-PHASE CHECKPOINT

A cell cycle checkpoint, typically activated in response to replication fork stalling or DNA damage, that blocks further origin initiation and stabilizes existing replication forks.

CLAMP LOADER

A protein that can open up a ring-shaped protein complex (such as the Mcm2–7 complex) and close it again so that the complex ends up encircling a DNA strand.

ENDOREDUPLICATION

A process in which cells gain more than the normal content of DNA, which typically occurs when cells embark on a second S phase before reducing their chromosome number by normal mitosis.

SCF-DEPENDENT MONOUBIQUITYLATION/POLYUBIQUITYLATION

Transfer of one (mono-) or several (poly-) ubiquitin molecules onto a protein which typically targets it for proteasome-mediated proteolysis.

SCF

The largest family of ubiquitin protein ligases; each is typically composed of a SKP1 homologue, a cullin, an F-box protein and a RING-domain protein, which ubiquitylates proteins, thereby marking them for proteasome-mediated proteolysis.

SKP2

A component of an SCF ubiquitin protein ligase, which provides an adaptor that links the F-box protein to the cullin.

CUL-4 UBIQUITIN LIGASE

A member of the cullin (SCF) ubiquitin ligase family, which ubiquitylates proteins thereby marking them for proteasome-mediated proteolysis.

COILED-COIL DOMAIN

A ubiquitous tertiary protein structure that consists of two to five α-helical strands coiled around one another.

CELL CYCLE CHECKPOINTS

Control pathways that ensure the integrity of the genome by interrupting cell-cycle progress if the DNA is damaged or replication forks stall. Kinases activated by these pathways can phosphorylate a range of other regulatory proteins (such as p53), which affect further cell-cycle progress or entry into apoptosis.

QUIESCENCE

Also known as G0, a state of reversible withdrawal from the cell cycle that can occur prior to entry into S phase.

SENESCENCE

A state of irreversible withdrawal from the cell cycle that occurs as a consequence of cell aging, typically thought to be triggered by erosion of telomere length.

E2F FAMILY

A family of transcription factors that plays an important part in regulating the transcription of key cell cycle genes in late G1 and S phase.

RB FAMILY

The retinoblastoma family, which includes the Rb protein itself and the related p107 and p130 proteins, which bind the E2F family of transcription factors and repress transcription.

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Blow, J., Dutta, A. Preventing re-replication of chromosomal DNA. Nat Rev Mol Cell Biol 6, 476–486 (2005). https://doi.org/10.1038/nrm1663

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