Regulation of the initiation of DNA replication in human cells
Section snippets
Preface for a general audience
DNA replication is targeted by chemotherapeutic drugs as cancer cells generally proliferate faster than most normal cells, and many cancers have acquired mutations that inactivate mechanisms that ensure genome stability. A precise understanding of the mechanisms that initiate DNA replication will allow the rational design of clinical trials of new agents and combinations that target DNA replication. At this time our understanding of the mechanisms that initiate DNA replication is largely
Complexity and timing of the initiation of DNA replication
The human genome evolved with mechanisms that assemble and activate the replicative helicase to initiate DNA unwinding and replication at ∼50,000 origins (indirect estimations showed ∼22,000 in HeLa [3], ∼50,000 in HeLa Kyoto [4]). The MCM2-7 hexamer is loaded onto DNA to license origins in G1 phase and CDC7 (CDC7 functions in a heterodimer with DBF4 and is also known as DDK for DBF4-dependent kinase) and CDK2 kinase activities initiate the assembly of CDC45, MCM2-7 and GINS and activation of
Replication complex assembly
The assembly of the replisome in human cells and all eukaryotic systems begins with origin licensing in the G1 phase of the cell cycle (Fig. 1a).
MCM loading on chromatin in G1 phase human cells requires ORC (Origin Recognition Complex) [34,35], CDT1 [36] and CDC6 [37] (Table 1), and these proteins need to be tightly regulated to prevent re-loading of the replicative helicase and re-replication in S phase [38]. However, the mechanisms preventing re-replication are quite different between
The signaling required for the initiation of DNA replication
The kinases CDC7 [85,86] and CDK2 [72] play critical roles in the assembly and activation of pre-replication complexes in human cells (Fig. 1c, Table 2) and as such their regulation determines the initiation of DNA replication. CMG helicase activation in yeast and human cells requires MCM phosphorylation by DDK. DBF4-dependent kinase (DDK) (Fig. 2c, d) is a complex of CDC7 and either DBF4 or DBF4b (DRF1) [87]. DBF4 or DBF4b is required for maximum CDC7 kinase activity and substrate recognition.
Regulation of replication initiation after DNA damage
Key elements of the DNA damage response include cell cycle arrest and inhibition of the initiation of DNA replication (Fig. 3). The regulation of DNA replication after DNA damage is a very complex process. While mild replication stress activates the initiation of replication from dormant origins, higher levels of damage completely block origin firing. Current models for this phenomenon suggests that low levels of ATR/CHK1 activity block the activation of replication in the new “replication
Conclusions and future directions
The initiation of DNA replication in humans is an extremely complex process, and caution is necessary when attempting to translate findings from yeast and Xenopus extract systems into mammalian cells. Major gaps in knowledge about origin firing in human cells include the mechanism though which the GINS complex and DNA polymerase epsilon are recruited to the MCM helicase and the mechanism that regulates DDK after DNA damage. Also, while the requirement for CDK2 and CDC7 activities for certain
Conflicts of interest
The authors declare that there are no conflicts of interest.
Author contributions
TM and CJB wrote the paper.
Acknowledgement
This work was supported by the NIH Grant RO1 CA204173.
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