DNA replication: Wakey wakey!

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The DNA-damage response (DDR) kinases ATR and CHK1 modulate the initiation of DNA replication by suppressing replication from secondary 'dormant' origin sites.

To maintain genomic stability, DNA replication must occur only once per cell cycle. DNA replication is 'licensed' in the preceding mitosis–early G1 phase, which involves the association of pre-replication complexes (preRCs) — comprising the ORC (origin recognition complex), CDC6, CDT1 and the MCM2–7 complex (the putative replicative helicase) — with origins of replication. The stoichiometry of DNA-associated MCM2–7 complexes compared with the number of origins used in a normal cell cycle is in $sim$ 20-fold excess. Julian Blow and colleagues now provide an explanation for this intriguing observation.

DNA replication is initiated from only a subset of preRCs, which fire in synchronous clusters at different times throughout S phase. But if replication can only be licensed prior to S phase, what happens to the DNA that is situated between two converging replication forks that become irreversibly stalled? Irreversible replication-fork stalling occurs as a result of DNA damage or collision with tightly associated protein complexes, and this activates the DNA-damage response (DDR) kinases ATR and CHK1, which orchestrate replication-fork stabilization, inhibition of late origin firing, DNA repair and cell-cycle checkpoint activation.

Using DNA fibre analyses, Blow and colleagues showed that active origins occurred every $sim$ 50 kb of DNA in human U2OS and MRC5 cells under normal culture conditions. After treatment with DNA replication inhibitors, such as hydroxyurea (HU), the density of active origins increased to one every $sim$ 25 kb and origin firing was less synchronous. Therefore, additional 'dormant' origins of replication appear to be fired once the primary forks are inhibited.

This is surprising because the DDR kinases prevent late origin firing and delay cell-cycle progression. The authors showed that depletion of CHK1 increased active replication origin density under normal conditions (one every $sim$ 40 kb) and that a further increase occurred after treatment with HU. This suggests that the DDR kinases suppress dormant origin firing under normal conditions, but dormant origins can be activated independently of DDR signalling in conditions of replicative stress.

Does the loading of excess MCM2–7 determine the abundance of dormant origins? Partial depletion of the MCM2–7 complex had no effect on normal cell cycles but prevented the increase in active origin density after treatment with HU. So, excess association of MCM2–7 is required for dormant origin firing under these conditions. Compared with control U2OS cells, partial depletion of MCM2–7 significantly reduced total replication rates in the presence of HU. This implies that dormant origins are required to rescue DNA replication when it is under stress. Consistently, MCM2–7-depleted cells were sensitive to inhibitors of DNA replication, indicating that this represents a new survival pathway that is required to maintain genomic stability.

The authors propose that dormant origins are activated passively, whereby slower replication rates that result from stress conditions increase the time in which a dormant origin can be fired. By contrast, the DDR partially suppresses the use of these origins under normal conditions, and they are inactivated by replication forks initiated from the primary origins. Whether DDR kinase-mediated suppression is different for early- and late-firing origins remains unclear.

Gemma K. Alderton

References

Ge, X. Q., Jackson, D. A. & Blow, J. J. Dormant origins licensed by excess Mcm2–7 are required for human cells to survive replicative stress. Genes Dev. 21, 3331–3341 (2007) | Article | PubMed |
Blow, J. J. & Dutta, A. Preventing re-replication of chromosomal DNA. Nature Rev. Mol. Cell Biol. 6, 476–486 (2005) | Article | PubMed |