A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication

Pasero, Philippe; Duncker, Bernard P.; Schwob, Étienne; Gasser, Susan M.

doi:10.1101/gad.13.16.2159

Cited by 115 publications

(138 citation statements)

References 71 publications

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“…Multiple mobility-shifted bands of huCdc7 after phosphorylation by Cdks indicate the presence of more than one phosphorylation sites on huCdc7. In yeasts, Cdc7-Dbf4 was reported to be associated with chromatin only during S phase (39). The possibility that cell cycle-dependent association and dissociation of Cdc7 kinase complex with chromatin may be regulated by phosphorylation by Cdks is now being examined.…”

Section: Discussionmentioning

confidence: 99%

Human Cdc7-related Kinase Complex

Masai¹,

Matsui²,

You³

et al. 2000

Journal of Biological Chemistry

142

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huCdc7 encodes a catalytic subunit for Saccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D., Seghezzi, W., Lees, E., Arai, K., and Masai, H. (1999) Mol. Cell. Biol. 19, 5083-5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathione S-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit. It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complex in vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.

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Section: Discussionmentioning

confidence: 99%

Human Cdc7-related Kinase Complex

Masai¹,

Matsui²,

You³

et al. 2000

Journal of Biological Chemistry

142

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show abstract

“…Both catalytic and regulatory subunits are localized in chromatin-enriched fractions, although they are not readily extractable by nuclease treatment, suggesting association of the Cdc7 kinases with nuclear structures (20). Association of Cdc7-Dbf4 proteins with chromatin during S phase was recently reported in budding yeast (39,40).…”

Section: Cell Cycle Regulation Of Cdc7 Kinase Activitymentioning

confidence: 99%

CDC7 kinase complex as a molecular switch for DNA replication

Masai¹

1999

Front Biosci

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“…Cdc7-Dbf4 is required for initiation of DNA replication (56, 57) and a likely Rad53 target to inhibit late origin firing, as activated Rad53 can phosphorylate Cdc7-Dbf4 and inhibit its kinase activity in vitro (58,59). In vivo, Dbf4 is phosphorylated and displaced from chromatin in a Rad53-dependent manner in response to HU to prevent Cdc45 and polymerase ␣ loading onto origins and new origin activation (60,61). Mcm2-7 are suggested to be direct targets of the Cdc7-Dbf4 kinase from in vitro studies (58,59,62), and it is speculated that their phosphorylation by Cdc7-Dbf4 may allow Mcm2-7 to bind Cdc45 required for replication initiation (42).…”

Section: Fha1 Functions In the Mrc1 Pathwaymentioning

confidence: 99%

Rad53 Kinase Activation-independent Replication Checkpoint Function of the N-terminal Forkhead-associated (FHA1) Domain

Pike

Tenis

Heierhorst

2004

Journal of Biological Chemistry

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Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9⌬ and rad17⌬) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1⌬). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G 2 /M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways.

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A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication

Cited by 115 publications

References 71 publications

Human Cdc7-related Kinase Complex

Human Cdc7-related Kinase Complex

CDC7 kinase complex as a molecular switch for DNA replication

Rad53 Kinase Activation-independent Replication Checkpoint Function of the N-terminal Forkhead-associated (FHA1) Domain

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