- Open Access
The role of CDK in the initiation step of DNA replication in eukaryotes
© Tanaka et al; licensee BioMed Central Ltd. 2007
- Received: 26 May 2007
- Accepted: 05 June 2007
- Published: 05 June 2007
Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle in eukaryotes. One of the major roles of CDK is to promote chromosomal DNA replication. However, how CDKs promote DNA replication has been a long-standing question, because all the essential CDK substrates in DNA replication have not been identified yet. Recently Sld2 and Sld3 were identified as essential substrates of CDKs in the initiation step of DNA replication in budding yeast. Moreover, bypass of their phosphorylations is sufficient to promote DNA replication. Phosphorylation of Sld2 and Sld3 by CDKs enhances the formation of complex(es) with a BRCT (BRCA1 C-Terminal)-containing replication protein, Dpb11. We further propose that multiple phosphorylation by CDKs controls this process in budding yeast. Even though Sld3 orthologues in multicellular eukaryotes have not been identified, similar complex formation and, therefore, a similar mechanism of initiation control might be employed in eukaryotes.
- Replication Origin
- Replication Protein
- BRCT Domain
- Replicative Helicase
- Multisite Phosphorylation
Chromosomal DNA replication in eukaryotes initiates from multiple replication origins. Activities of individual origins are regulated as follows: The six-subunit origin recognition complex (Orc) binds to replication origins throughout the cell cycle in yeast cells, and the putative replicative helicase Mcm (Mcm2-7 complex) is recruited onto Orc-bound origins with the aid of Cdc6 and Cdt1 to form the pre-replicative complex (pre-RC), from late M to G1 phase when CDK activity is low. Once both CDK and Dbf4-dependent protein kinase (DDK/Cdc7), another protein kinase essential for DNA replication, are activated, many replication proteins including replicative DNA polymerases, α, δ and ε are loaded onto the pre-RC formed origins. Then origin DNA is unwound and replication forks are formed to synthesize DNA (see reviews; [8, 9]). For DNA replication after the initiation, both CDK and DDK are dispensable [2, 10, 11], our unpublished results].
We previously showed that budding-yeast Sld2 is an essential CDK target for DNA replication . The sld2 mutation was originally isolated as one of sld (synthetically lethal with dpb11-1) . The Sld2 protein has a cluster of 11 CDK phosphorylation motifs (Ser/Thr-Pro). At one of these motifs, phosphorylation of Thr84 promotes an essential complex formation between Sld2 and another replication protein, Dpb11, while Thr84-phosphorylation requires other phosphorylations in Sld2 (see below) . Dpb11 has two pairs of tandem BRCT (BRCA1 C-Terminal) domains, which is known as a phosphopeptide-binding domain [14, 15]. C-terminal pair of BRCT domains in Dpb11 binds to phosphoThr84-Sld2 . Dpb11 and Sld2 associate with replication origins when Cdk1 (Cdc28 in budding yeast) is activated , our unpublished results]. However, phosphorylation of Sld2 alone does not promote initiation of DNA replication. A phosphomimetic form of Sld2 constructed by substituting aspartic acid for serine or threonine residue in CDK phosphorylation motif could support cell growth but Cdk1 was still required for initiation . This strongly suggests the existence of other targets of S-phase CDK at the initiation step of DNA replication.
Actually, we and others found another essential CDK target, Sld3 [5, 6]. The sld3 mutation was also isolated by the 'sld' screening . Of 12 CDK phosphorylation motifs in Sld3, phosphorylations at Thr600 and Ser622 are crucial for binding Sld3 to N-terminal pair of tandem BRCT domains in Dpb11. Simultaneous alanine substitutions (Sld3-2A) and any combination of aspartic acid and glutamic acid substitutions for these residues confer cell lethality and defects of DNA replication, suggesting that phosphorylations of Thr600 and Ser622 are essential for cell growth and DNA replication and aspartic acid and glutamic acid residues do not mimic a phosphorylated residue at these sites. However, there are three ways to restore growth of sld3-2A mutant cells. First, multicopy DPB11 suppresses lethality caused by sld3-2A . Overexpression of binding partners often suppresses a mutation occurring in the other subunit. This is the case for Dpb11 and Sld3. Second, the JET1-1 mutation (J umping E ssentiality of CDK with sld2(T wo)-11D to initiate DNA replication) restores the growth defect . JET1-1 was isolated as a mutation, which induces untimely DNA replication when the phosphomimetic Sld2 (Sld2-11D) was expressed. The JET1-1 mutation occurred in the CDC45 gene, whose product binds to Sld3. However, Cdc45 itself is not a target of Cdk1 in the initiation, because disruption of all the CDK phosphorylation motifs in Cdc45 does not show any defect in DNA replication nor cell growth. Jet1-1 seems to enhance the interaction between Sld3 and Dpb11 through the interaction between Cdc45 and Sld3 . Third, the fusion protein between Sld3-2A and C-terminal half of Dpb11 (SD fusion) can replace Sld3 and Dpb11 simultaneously because N-terminal pair of Dpb11 functions as the binding site to Sld3 and SD fusion functions as Sld3-Dpb11 complex. These results imply that phosphorylation-dependent interaction between Dpb11 and Sld3 is CDK-regulated essential step for the initiation of DNA replication.
We combined JET1-1, multicopy DPB11 and the sld2-D mutation while Zegerman and Diffley combined the SD fusion and the sld2-D mutation [5, 6]. In any case, the combinatory strains initiated DNA synthesis even when lacking S-phase CDK activities. DNA synthesis observed in the absence of CDK activity reflects DNA replication by several criteria. In normal DNA replication, two protein kinases, CDK and DDK, activate DNA replication. Both combinatory strains require DDK activity to initiate DNA synthesis, indicating that the cells only bypass CDK requirement in the initiation. Furthermore, semi-conservative DNA synthesis initiates from known replication origins in a pre-RC-dependent manner with proper replication fork complex, CMG (Cdc45-Mcm-GINS) in JET1-1 sld2-D mutant cells lacking CDK activity (DNA synthesis in SD fusion strain has not been characterized in detail). In any combinatory strains, DNA replication induced in G1 cells is not efficient as that in CDK-driven DNA replication. Nonetheless, these data strongly suggest that phosphorylation of Sld2 and Sld3 by Cdk1 are the minimal requirement for the initiation of DNA replication although we cannot rule out the formal possibility that the SD fusion and JET1 bypass not only Sld3 phosphorylation but also other phosphorylations.
While phosphorylation-dependent interactions between Sld2, Sld3 and Dpb11 appear to be essential to initiate DNA replication, how these interactions facilitate initiation is still obscure. A novel complex, pre-Loading Complex (pre-LC) containing Polε, GINS, Dpb11 and Sld2 was detected in the cells treated with a cross-linking reagent and this complex formation depends on CDK but not DDK nor pre-RC (our unpublished results). On the other hand, Sld3, together with Cdc45, associates with pre-RC-formed early-firing origins even in G1 phase . Thus, when Cdk1 is activated, Sld3 on origins is phosphorylated and binds to Dpb11, which might be a component of the pre-LC (Fig. 1). While we do not know whether Dpb11 binds to Sld2 and Sld3 in a sequential or random manner, and whether a single Dpb11 molecule binds to Sld3 and Sld2 simultaneously, these interaction may contribute to association between proteins bound to Sld2, Sld3 and Dpb11, such as GINS, Cdc45 and Mcm. Recent studies have identified complexes containing Cdc45, Mcm and GINS in Xenopus, budding yeast, and Drosophila, which are suggested to work at replication forks [18–20]. Moreover, the CMG complex composed of these three factors from Drosophila shows helicase activity, suggesting that it works as a replicative helicase . Thus, we suggest CDK-phosphorylation dependent interactions between Dpb11, Sld2 and Sld3 may play a role in the formation of CMG complex.
The phosphorylation-dependent interaction between Dpb11, Sld2 and Sld3 may be an essential mechanistic requirement to promote the initiation of DNA replication. It is also conceivable that these interactions monitor cellular CDK activity to couple DNA replication with other cell cycle events. We will discuss the interaction between Dpb11 and Sld2 from our viewpoint.
The peptide corresponding to residues 79–107 of Sld2 binds to the C-terminal pair of tandem BRCT domains in a Thr84-phosphorylation dependent manner. However, disruption of other CDK phosphorylation sites of Sld2 abolishes the interaction between Dpb11 and Sld2 . This is because phosphorylation at Thr84 requires prior phosphorylation of other sites by Cdk1. Thus, phosphorylation of Thr84 occurs later than other phosphorylations sites and consequently multisite phosphorylation set a high threshold for CDK activity to form complex between Dpb11 and Sld2. Conversion of the CDK phosphorylation motif, Thr84-Pro, to PIKK (PI 3 k inase related k inase) phosphorylation motif, Thr84-Gln, results in phosphorylation of Thr84 by DNA-dependent protein kinase, one of PIKK, and this phosphorylation depends on pre-phosphorylation by CDK. We thus argue that phosphorylation renders Thr84 accessible to DNA-dependent kinase in the Thr-Gln construct and to Cdk1 in the original construct, probably by phosphorylation-dependent conformational change (Fig. 2).
The high threshold of CDK activity may prevent premature replication. When Cdk1 is activated and DNA replication initiates, the pre-RC is converted to post-replicative complex (post-RC) composed of Orc at the replication origins. Active Cdk1 phosphorylates the pre-RC components and inhibits reassembly of pre-RC to prevent reinitiation (reviewed by ). The high threshold of CDK activity for Thr84 phosphorylation ensures complete phosphorylation of the pre-RC components before DNA replication initiates.
The Sld3 protein has 12 CDK phosphorylation motifs. Two of them, Thr600 and Ser622, are responsible for CDK phosphorylation-dependent interaction between Dpb11 and Sld3 [5, 6]. Simultaneous alanine substitution at all the CDK phosphorylation motifs except Thr600 and Ser622 does not affect cell growth, suggesting that phosphorylation of Sld3 does not affect phosphorylation of Thr600 and Ser622, unlike in Sld2. On the other hand, efficient binding of Sld3 to N-terminal pair of BRCT domains in Dpb11 requires simultaneous phosphorylations of Thr600 and Ser622, which may require high activity of Cdk1. Further investigation will reveal the precise regulation of phosphorylation-dependent interaction between Sld3 and Dpb11 and how Sld2 and Sld3 bind to Dpb11, for example sequential or random.
4x BRCT. N-terminal and C-terminal pair binds phosphorylated Sld3 and phosphorylated Sld2, respectively. [6, 12, 14]
4x BRCT. N-terminal pair is shown to bind Drc1. [25, 26]
6x BRCT. Required for DNA replication. MMS sensitibity by RNAi feeding. SUMO modification? 
7x BRCT. Involvement in DNA replication is suggested. [28, 29]
8x BRCT. Functions in DNA replication and DNA replication or damage checkpoints. [30–32]
8x BRCT. Originally isolated as topoisomerase-binding protein. Functions in DNA replication and DNA replication or damage checkpoints. [33–35]
Phosphorylation at T84 by CDK is essential for initiation.
Phosphorylated Sld2 binds Dpb11 C-terminus.
Alanine-substitution mutant is lethal. [7, 12]
Phosphorylation by CDK is essential for initiation.
Alanine-substitution mutant is lethal. 
N-terminal portion show similarity to Sld2. C-terminal portion has RecQ helicase motif. It binds Cut5 and is required for DNA replication. 
H. sapiens M.musculus
N-terminal portion show similarity to Sld2. C-terminal portion has RecQ helicase motif. It is essential for cell proliferation.
Rothmund-Thomson syndrome and Rapadilino syndrome cells have mutaions in this gene. 
Binds to Cdc45, GINS and Dpb11. Phosphorylation at T600 and S622 by CDK is essential for initiation.
Phosphorylated Sld3 binds Dpb11 N-terminus. Alanine-substitution mutant is lethal. [5, 6]
Required for initiation (Cdc45 loading). Chromatin loading of Sld3 depends on DDK but not CDK. 
In budding yeast Cdk1 promotes chromosomal DNA replication by phosphorylating two replication proteins, Sld2 and Sld3. Phosphorylated form of Sld2 and Sld3 bind to BRCT-containing protein, Dpb11 and these interactions are essential for initiating DNA replication. Bypassing these phosphorylation-dependent interactions by various ways promotes DNA replication in the absence of CDK activity. Thus, we conclude that CDK-dependent phosphorylations of Sld2 and Sld3 and consequently their interactions with Dpb11 are minimal requirements for CDK-dependent activation of the initiation of DNA replication. Furthermore, multiple phosphorylation of Sld2, at least, contributes to fine-tuning of this step. Although Dpb11, Sld2 and Sld3 are not well conserved in multi-cellular organisms, CDK may regulate the initiation step of DNA replication in a similar manner, that is, phosphorylation-dependent interactions of replication proteins.
The study was partly supported by grants to H.A and S. T. from Ministry of Education, Culture, Sports, Science and Technology, Japan.
- D'Urso G, Marraccino RL, Marshak DR, Roberts JM: Cell cycle control of DNA replication by a homologue from human cells of the p34cdc2 protein kinase. Science 1990,250(4982):786–791. 10.1126/science.2173140PubMedView ArticleGoogle Scholar
- Blow JJ, Nurse P: A cdc2-like protein is involved in the initiation of DNA replication in Xenopus egg extracts. Cell 1990,62(5):855–862. 10.1016/0092-8674(90)90261-CPubMedView ArticleGoogle Scholar
- Hartwell LH, Culotti J, Pringle JR, Reid BJ: Genetic control of the cell division cycle in yeast. Science 1974,183(120):46–51. 10.1126/science.183.4120.46PubMedView ArticleGoogle Scholar
- Schwob E, Nasmyth K: CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae. Genes Dev 1993,7(7A):1160–1175. 10.1101/gad.7.7a.1160PubMedView ArticleGoogle Scholar
- Zegerman P, Diffley JF: Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. Nature 2007,445(7125):281–285. 10.1038/nature05432PubMedView ArticleGoogle Scholar
- Tanaka S, Umemori T, Hirai K, Muramatsu S, Kamimura Y, Araki H: CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast. Nature 2007,445(7125):328–332. 10.1038/nature05465PubMedView ArticleGoogle Scholar
- Tak YS, Tanaka Y, Endo S, Kamimura Y, Araki H: A CDK-catalysed regulatory phosphorylation for formation of the DNA replication complex Sld2-Dpb11. Embo J 2006,25(9):1987–1996. 10.1038/sj.emboj.7601075PubMed CentralPubMedView ArticleGoogle Scholar
- Diffley JF: Regulation of early events in chromosome replication. Curr Biol 2004,14(18):R778–86. 10.1016/j.cub.2004.09.019PubMedView ArticleGoogle Scholar
- Bell SP, Dutta A: DNA replication in eukaryotic cells. Annu Rev Biochem 2002, 71: 333–374. 10.1146/annurev.biochem.71.110601.135425PubMedView ArticleGoogle Scholar
- Bousset K, Diffley JF: The Cdc7 protein kinase is required for origin firing during S phase. Genes Dev 1998,12(4):480–490.PubMed CentralPubMedView ArticleGoogle Scholar
- Donaldson AD, Fangman WL, Brewer BJ: Cdc7 is required throughout the yeast S phase to activate replication origins. Genes Dev 1998,12(4):491–501.PubMed CentralPubMedView ArticleGoogle Scholar
- Masumoto H, Muramatsu S, Kamimura Y, Araki H: S-Cdk-dependent phosphorylation of Sld2 essential for chromosomal DNA replication in budding yeast. Nature 2002,415(6872):651–655. [http://www.nature.com/nature/journal/v415/n6872/full/nature713.html] 10.1038/nature713PubMedView ArticleGoogle Scholar
- Kamimura Y, Masumoto H, Sugino A, Araki H: Sld2, which interacts with Dpb11 in Saccharomyces cerevisiae, is required for chromosomal DNA replication. Mol Cell Biol 1998,18(10):6102–6109.PubMed CentralPubMedView ArticleGoogle Scholar
- Araki H, Leem SH, Phongdara A, Sugino A: Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint. Proc Natl Acad Sci U S A 1995,92(25):11791–11795. 10.1073/pnas.92.25.11791PubMed CentralPubMedView ArticleGoogle Scholar
- Glover JN, Williams RS, Lee MS: Interactions between BRCT repeats and phosphoproteins: tangled up in two. Trends Biochem Sci 2004,29(11):579–585. 10.1016/j.tibs.2004.09.010PubMedView ArticleGoogle Scholar
- Masumoto H, Sugino A, Araki H: Dpb11 controls the association between DNA polymerases alpha and epsilon and the autonomously replicating sequence region of budding yeast. Mol Cell Biol 2000,20(8):2809–2817. 10.1128/MCB.20.8.2809-2817.2000PubMed CentralPubMedView ArticleGoogle Scholar
- Kamimura Y, Tak YS, Sugino A, Araki H: Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae. Embo J 2001,20(8):2097–2107. [http://www.nature.com/emboj/journal/v20/n8/full/7593709a.html] 10.1093/emboj/20.8.2097PubMed CentralPubMedView ArticleGoogle Scholar
- Pacek M, Tutter AV, Kubota Y, Takisawa H, Walter JC: Localization of MCM2–7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication. Mol Cell 2006,21(4):581–587. 10.1016/j.molcel.2006.01.030PubMedView ArticleGoogle Scholar
- Moyer SE, Lewis PW, Botchan MR: Isolation of the Cdc45/Mcm2–7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc Natl Acad Sci U S A 2006,103(27):10236–10241. 10.1073/pnas.0602400103PubMed CentralPubMedView ArticleGoogle Scholar
- Gambus A, Jones RC, Sanchez-Diaz A, Kanemaki M, van Deursen F, Edmondson RD, Labib K: GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nat Cell Biol 2006,8(4):358–366. [http://www.nature.com/ncb/journal/v8/n4/full/ncb1382.html] 10.1038/ncb1382PubMedView ArticleGoogle Scholar
- Arias EE, Walter JC: Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. Genes Dev 2007,21(5):497–518. 10.1101/gad.1508907PubMedView ArticleGoogle Scholar
- Garcia V, Furuya K, Carr AM: Identification and functional analysis of TopBP1 and its homologs. DNA Repair (Amst) 2005,4(11):1227–1239. 10.1016/j.dnarep.2005.04.001View ArticleGoogle Scholar
- Matsuno K, Kumano M, Kubota Y, Hashimoto Y, Takisawa H: The N-terminal noncatalytic region of Xenopus RecQ4 is required for chromatin binding of DNA polymerase alpha in the initiation of DNA replication. Mol Cell Biol 2006,26(13):4843–4852. 10.1128/MCB.02267-05PubMed CentralPubMedView ArticleGoogle Scholar
- Nakajima R, Masukata H: SpSld3 is required for loading and maintenance of SpCdc45 on chromatin in DNA replication in fission yeast. Mol Biol Cell 2002,13(5):1462–1472. 10.1091/mbc.02-01-0006PubMed CentralPubMedView ArticleGoogle Scholar
- Saka Y, Fantes P, Yanagida M: Coupling of DNA replication and mitosis by fission yeast rad4/cut5. J Cell Sci Suppl 1994, 18: 57–61.PubMedView ArticleGoogle Scholar
- Noguchi E, Shanahan P, Noguchi C, Russell P: CDK phosphorylation of Drc1 regulates DNA replication in fission yeast. Curr Biol 2002,12(7):599–605. 10.1016/S0960-9822(02)00739-XPubMedView ArticleGoogle Scholar
- Holway AH, Hung C, Michael WM: Systematic, RNA-interference-mediated identification of mus-101 modifier genes in Caenorhabditis elegans. Genetics 2005,169(3):1451–1460. 10.1534/genetics.104.036137PubMed CentralPubMedView ArticleGoogle Scholar
- Boyd JB, Golino MD, Nguyen TD, Green MM: Isolation and characterization of X-linked mutants of Drosophila melanogaster which are sensitive to mutagens. Genetics 1976,84(3):485–506.PubMed CentralPubMedGoogle Scholar
- Yamamoto RR, Axton JM, Yamamoto Y, Saunders RD, Glover DM, Henderson DS: The Drosophila mus101 gene, which links DNA repair, replication and condensation of heterochromatin in mitosis, encodes a protein with seven BRCA1 C-terminus domains. Genetics 2000,156(2):711–721.PubMed CentralPubMedGoogle Scholar
- Van Hatten RA, Tutter AV, Holway AH, Khederian AM, Walter JC, Michael WM: The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication. J Cell Biol 2002,159(4):541–547. 10.1083/jcb.200207090PubMed CentralPubMedView ArticleGoogle Scholar
- Parrilla-Castellar ER, Karnitz LM: Cut5 is required for the binding of Atr and DNA polymerase alpha to genotoxin-damaged chromatin. J Biol Chem 2003,278(46):45507–45511. 10.1074/jbc.C300418200PubMedView ArticleGoogle Scholar
- Hashimoto Y, Takisawa H: Xenopus Cut5 is essential for a CDK-dependent process in the initiation of DNA replication. Embo J 2003,22(10):2526–2535. [http://www.nature.com/emboj/journal/v22/n10/full/7590649a.html] 10.1093/emboj/cdg238PubMed CentralPubMedView ArticleGoogle Scholar
- Yamane K, Kawabata M, Tsuruo T: A DNA-topoisomerase-II-binding protein with eight repeating regions similar to DNA-repair enzymes and to a cell-cycle regulator. Eur J Biochem 1997,250(3):794–799. 10.1111/j.1432-1033.1997.00794.xPubMedView ArticleGoogle Scholar
- Makiniemi M, Hillukkala T, Tuusa J, Reini K, Vaara M, Huang D, Pospiech H, Majuri I, Westerling T, Makela TP, Syvaoja JE: BRCT domain-containing protein TopBP1 functions in DNA replication and damage response. J Biol Chem 2001,276(32):30399–30406. 10.1074/jbc.M102245200PubMedView ArticleGoogle Scholar
- Jeon Y, Lee KY, Ko MJ, Lee YS, Kang S, Hwang DS: Human TopBP1 Participates in Cyclin E/CDK2 Activation and Preinitiation Complex Assembly during G1/S Transition. J Biol Chem 2007,282(20):14882–14890. 10.1074/jbc.M609116200PubMedView ArticleGoogle Scholar
- Sangrithi MN, Bernal JA, Madine M, Philpott A, Lee J, Dunphy WG, Venkitaraman AR: Initiation of DNA replication requires the RECQL4 protein mutated in Rothmund-Thomson syndrome. Cell 2005,121(6):887–898. 10.1016/j.cell.2005.05.015PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.