ReviewEukaryotic DNA replication: from pre-replication complex to initiation complex
Introduction
The replication of chromosomal DNA is central for the duplication of a cell. In eukaryotes, a conserved mechanism operates to restrict DNA replication to only once per cell cycle. In this review, we will call this mechanism the ‘licensing’ of DNA replication. Recent studies have shed light on the licensing mechanisms, in particular, the mechanisms for the formation of the pre-replicative complex (pre-RC) at origins (which establishes the licensed state of chromatin), the conversion of pre-RC into an initiation complex that leads to the initiation of DNA synthesis, and the prevention of re-replication. In the conversion process, two kinds of S phase promoting kinases, conserved from yeast to human, are involved [1]. The role of these kinases in the initiation reaction has not yet been precisely determined, but now is the right time to propose a unified view for the mechanism of initiation of eukaryotic DNA replication. In this review, we will focus on progress in understanding the initiation machinery in the past year. Previous studies were excellently reviewed two years ago [2] and readers are also encouraged to refer to reviews on the other aspects of DNA replication, such as the regulation of replication origin activation [3], parallels between eukaryotes and prokaryotes [4] and eukaryotic replication origins [5], [6].
Section snippets
Pre-RC—MCM loaded chromatin
In this review, we would like to define the pre-RC as the protein complex formed at origins that establishes the licensed state. This definition is in accord with the original definition of the pre-replicative state, which is detected as G1 phase specific footprinting over replication origins [7]. In this context, the formation of pre-RCs can be described as the sequential assembly of the origin recognition complex (ORC), Cdc6/cdc18 and MCM onto origins. Recent studies with the fission yeast [8]
Cdc45—a component of the pre-RC?
In addition to MCM, Cdc45 is essential for initiating DNA replication [2], [17]. Recent studies have clearly shown that MCM is critical for the loading of Cdc45 onto chromatin and a complex containing Cdc45 and MCM was formed at the onset of S phase [18], [19. These results suggest that Cdc45 targets MCM, which has been loaded onto chromatin as a component of the pre-RC. A chromatin immunoprecipitation (CHIP) assay of Cdc45 and MCM further support the view that such a protein complex is formed
Two protein kinases triggering DNA replication
At the onset of S phase, the pre-RC must be activated by two S phase specific kinases in order to form an initiation complex at an origin. One is the Cdc7–Dbf4 kinase called DDK (Dbf4-dependent kinase) [24] and the other is CDK. Chromatin-binding assays of Cdc45 in yeast and Xenopus show that one of the downstream events of CDK action is the loading of Cdc45 onto chromatin [17], [18]. In mammalian cells, it has also been suggested that cdk2 activity is required for the loading of Cdc45 onto
CDC45 as a key protein for establishing the initiation complex
In previous sections, we have discussed the consequence of S phase kinase action as the formation of MCM–Cdc45 complex onto origins. The loading of Cdc45 onto chromatin is critical for loading various replication proteins, including DNA polymerase α, DNA polymerase ε, replication protein A (RPA) and proliferating cell nuclear antigen (PCNA) onto chromatin [19, [21, [22, [41. An obvious question is what happens to DNA after the loading of Cdc45 onto chromatin. Recent studies with the Xenopus
MCM—a replicative helicase?
MCM can now be considered a good candidate for the replicative helicase. Recent studies showed that recombinant Mcm4–Mcm6–Mcm7 complexes of mammalian and fission yeast have intrinsically weak helicase activities in vitro [44, [45. Studies with an archaeal MCM protein suggest that the oligomer of the protein has helicase activity [46, [47, [48. In addition, a recent study with an MCM degron mutant indicates that MCM is required for both the initiation and the elongation stages of DNA replication
DNA polymerase ε—only for DNA synthesis?
Previous studies on the SV40 DNA replication system provide us with a framework for understanding the role of DNA polymerases in replication [51]. Upon the unwinding of DNA by large T antigen, DNA polymerase α is recruited onto unwound DNA through its physical interaction with RPA and large T antigen. The replication reaction is primed by DNA polymerase α, which synthesizes a short stretch of DNA following primer RNA synthesis. Replication factor C (RFC), a clamp loader, then recognizes the 3′
New faces in the initiation reaction
In addition to Cdc45, Dpb11 plays an important role in recruiting DNA polymerases onto origins [52]. DPB11 was isolated as a multicopy suppressor of dpb2 (which encodes the second largest subunit of polymerase ε) mutants and it also suppresses mutations in pol2 (which encodes the catalytic subunit of DNA polymerase ε). It is also involved in both the replication and checkpoint functions of DNA polymerase ε [55]. cut5 of fission yeast, a putative homolog of DPB11, is also required for S phase
Conclusions
The licensing of replication has been discussed in terms of the control of pre-RC formation and the pre-RC has now been recognized as a major target of S phase kinases. Recent studies have further shown that Cdc45 plays a central role in the activation of the pre-RC. Emerging evidence suggests that MCM–Cdc45 is a component of an active helicase, which also leads to the formation of an initiation complex on unwound DNA. This notion urgently requires experimental proof. Further, recent studies on
Acknowledgements
Current work by the authors is supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan. We are very grateful to JJ Blow for helpful discussion and comments, to H Araki and J Diffley for helpful discussion and J Diffley and J Walter for permitting us to cite unpublished observations.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
of special interest
of outstanding interest
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2015, BiochimieCitation Excerpt :Origin recognition complexes (ORCs) recognize the origins of replication, a sequence possibly AT-rich, possessing strand asymmetry and oligo-A tracks (leading to DNA curvature) [22]. Two kinds of kinases are involved in the conversion of pre-RC into the initiation complex, namely Cdc7-Dbf4 kinase (Dbf4-dependent kinase or DDK) and CDK, where the role of both is to load Cdc45 onto chromatin [8,22,26,29,30]. In this process Cdc6 is destabilized and MCM10 is recruited into the pre-RC [22].