The role of CDK in the initiation step of DNA replication in eukaryotes

Tanaka, Seiji; Tak, Yon-Soo; Araki, Hideki

doi:10.1186/1747-1028-2-16

Cited by 62 publications

(49 citation statements)

References 36 publications

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“…TopBP1 is involved in DNA replication and checkpoints, as observed for its yeast counterparts (Garcia et al 2005;Walter and Araki 2006;Tanaka et al 2007a;Labib 2010;Tanaka and Araki 2010). Although yeast Dpb11/Cut5 has four BRCT domains, metazoan TopBP1s have multiple BRCTs (more than six).…”

Section: Topbp1mentioning

confidence: 99%

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

Tanaka

Araki

2013

Cold Spring Harbor Perspectives in Biology

156

146

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Many replication proteins assemble on the pre-RC-formed replication origins and constitute the pre-initiation complex ( pre-IC). This complex formation facilitates the conversion of Mcm2-7 in the pre-RC to an active DNA helicase, the Cdc45-Mcm-GINS (CMG) complex. Two protein kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), work to complete the formation of the pre-IC. Each kinase is responsible for a distinct step of the process in yeast; Cdc45 associates with origins in a DDK-dependent manner, whereas the association of GINS with origins depends on CDK. These associations with origins also require specific initiation proteins: Sld3 for Cdc45; and Dpb11, Sld2, and Sld3 for GINS. Functional homologs of these proteins exist in metazoa, although pre-IC formation cannot be separated by requirement of DDK and CDK because of experimental limitations. Once the replicative helicase is activated, the origin DNA is unwound, and bidirectional replication forks are established.

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Section: Topbp1mentioning

confidence: 99%

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

Tanaka

Araki

2013

Cold Spring Harbor Perspectives in Biology

156

146

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“…Dpb11 is evolutionarily conserved (10,11). The functions and overall structures of Dpb11 of budding yeast and of its ortholog in fission yeast, Cut5, are similar (12, 13) (see Fig.…”

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confidence: 95%

Efficient Initiation of DNA Replication in Eukaryotes Requires Dpb11/TopBP1-GINS Interaction

Tanaka

Komeda

Umemori

et al. 2013

Molecular and Cellular Biology

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c Dpb11/Cut5/TopBP1 is evolutionarily conserved and is essential for the initiation of DNA replication in eukaryotes. The Dpb11 of the budding yeast Saccharomyces cerevisiae has four BRCT domains (BRCT1 to -4). The N-terminal pair (BRCT1 and -2) and the C-terminal pair (BRCT3 and -4) bind to cyclin-dependent kinase (CDK)-phosphorylated Sld3 and Sld2, respectively. These phosphorylation-dependent interactions trigger the initiation of DNA replication. BRCT1 and -2 and BRCT3 and -4 of Dpb11 are separated by a short stretch of ϳ100 amino acids. It is unknown whether this inter-BRCT region functions in DNA replication. Here, we showed that the inter-BRCT region is a GINS interaction domain that is essential for cell growth and that mutations in this domain cause replication defects in budding yeast. We found the corresponding region in the vertebrate ortholog, TopBP1, and showed that the corresponding region also interacts with GINS and is required for efficient DNA replication. We propose that the inter-BRCT region of Dpb11 is a functionally conserved GINS interaction domain that is important for the initiation of DNA replication in eukaryotes. Chromosomal DNA replication in eukaryotes occurs as a twostep reaction whose steps are separated temporally in the cell cycle (see reviews in references 1 and 2). In the first reaction, called licensing, the core component of the replicative helicase, Mcm2-7, is loaded onto replication origins to assemble the prereplicative complexes (pre-RCs) in the G 1 phase of the cell cycle. At this point, the Mcm2-7 helicase is inactive in the pre-RC. In the following S phase, the replicative helicase is activated by the functions of two protein kinases-cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK)-and the replication fork is established (initiation reaction). In the budding yeast Saccharomyces cerevisiae, Sld2 and Sld3 are essential targets of CDK. CDK phosphorylation of Sld2 and Sld3 promotes a phosphodependent interaction with the tandem BRCT repeats of another replication factor called Dpb11 (3-6). These interactions promote the recruitment of GINS onto origins via the preloading complex (pre-LC), which consists of Dpb11, Sld2, DNA polymerase ε (Pol ε), and GINS. The pre-LC is an important intermediate in the subsequent formation of the active replicative helicase, which is called the CMG (Cdc45-Mcm2-7-GINS) complex, and the establishment of the replication fork (7-9). Therefore, Dpb11 is a key mediator of the formation of the new complex on replication origins and plays a crucial role in the initiation of DNA replication.Dpb11 is evolutionarily conserved (10, 11). The functions and overall structures of Dpb11 of budding yeast and of its ortholog in fission yeast, Cut5, are similar (12, 13) (see Fig. S1 in the supplemental material). The vertebrate ortholog, TopBP1, is also a BRCT-containing protein (14), the N-terminal region of which contains BRCT1 and -2, which interacts with phosphorylated treslin, an ortholog of vertebrate Sld3 (15,16). Although TopBP1 is also c...

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“…The S phase cyclin-dependent kinase also activates yeast origins of replication (3)(4)(5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6).…”

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Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

Bruck

Kaplan

2009

Journal of Biological Chemistry

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The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2⌬); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3)(4)(5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK) 2 acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7-10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11)(12)(13)(14)(15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16) In budding yeast, Cdc7 is present at high levels in G 1 and S phase, whereas Dbf4 levels peak in S phase (18,21,22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21-24). However, a mechanistic analysis of how Dbf4 activates Cdc7has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19,20,28) In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 r...

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The role of CDK in the initiation step of DNA replication in eukaryotes

Cited by 62 publications

References 36 publications

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

Efficient Initiation of DNA Replication in Eukaryotes Requires Dpb11/TopBP1-GINS Interaction

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

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