Suppression of targeting of Dbf4-dependent kinase to pre-replicative complex in G0 nuclei.

Intact G0 nuclei isolated from quiescent cells are not capable of DNA replication in interphase Xenopus egg extracts, which allow efficient replication of permeabilized G0 nuclei. Previous studies have shown multiple control mechanisms for maintaining the quiescent state, but DNA replication inhibition of intact G0 nuclei in the extracts remains poorly understood. Here, we showed that pre-RC is assembled on chromatin, but its activation is inhibited after incubating G0 nuclei isolated from quiescent NIH3T3 cells in the extracts. Concomitant with the inhibition of replication, Mcm4 phosphorylation mediated by Dbf4-dependent kinase (DDK) as well as chromatin binding of DDK is suppressed in G0 nuclei without affecting the nuclear transport of DDK. We further found that the nuclear extracts of G0 but not proliferating cells inhibit the binding of recombinant DDK to pre-RC assembled plasmids. In addition, we observed rapid activation of checkpoint kinases after incubating G0 nuclei in the egg extracts. However, specific inhibitors of ATR/ATM are unable to promote DNA replication in G0 nuclei in the egg extracts. We suggest that a novel inhibitory mechanism is functional to prevent the targeting of DDK to pre-RC in G0 nuclei, thereby suppressing DNA replication in Xenopus egg extracts.

DDK promotes DNA replication initiation: Mechanistic and structural insights.

DNA replication initiation in eukaryotes is tightly regulated through two cell-cycle specific processes, replication licensing to install inactive minichromosome maintenance (MCM) double-hexamers (DH) on origins in early G1 phase and origin firing to assemble and activate Cdc45-Mcm2-7-GINS (CMG) helicases upon S phase entry. Two kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are responsible for driving the association of replication factors with the MCM-DH to form CMG helicases for origin melting and DNA unwinding and eventually replisomes for bi-directional DNA synthesis. In recent years, cryo-electron microscopy studies have generated a collection of structural snapshots for the stepwise assembly and remodeling of the replication initiation machineries, creating a framework for understanding the regulation of this fundamental process at a molecular level. Very recent progress is the structural characterization of the elusive MCM-DH-DDK complex, which provides insights into mechanisms of kinase activation, substrate recognition and selection, as well as molecular role of DDK-mediated MCM-DH phosphorylation in helicase activation.

Dbf4 Zn-Finger Motif Is Specifically Required for Stimulation of Ctf19-Activated Origins in Saccharomyces cerevisiae.

Eukaryotic genomes are replicated in spatiotemporal patterns that are stereotypical for individual genomes and developmental profiles. In the model system Saccharomyces cerevisiae, two primary mechanisms determine the preferential activation of replication origins during early S phase, thereby largely defining the consequent replication profiles of these cells. Both mechanisms are thought to act through specific recruitment of a rate-limiting initiation factor, Dbf4-dependent kinase (DDK), to a subset of licensed replication origins. Fkh1/2 is responsible for stimulation of most early-firing origins, except for centromere (CEN)-proximal origins that recruit DDK via the kinetochore protein Ctf19, which is required for their early firing. The C-terminus of Dbf4 has been implicated in its recruitment to origins via both the Fkh1/2 and Ctf19 mechanisms. Here, we show that the Zn-finger motif within the C-terminus is specifically required for Dbf4 recruitment to CENs to stimulate CEN-proximal/Ctf19-dependent origins, whereas stimulation of origins via the Fkh1/2 pathway remains largely intact. These findings re-open the question of exactly how Fkh1/2 and DDK act together to stimulate replication origin initiation.

Dbf4-Cdc7 (DDK) Inhibitor PHA-767491 Displays Potent Anti-Proliferative Effects via Crosstalk with the CDK2-RB-E2F Pathway.

Precise regulation of DNA replication complex assembly requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) activities to activate the replicative helicase complex and initiate DNA replication. Chemical probes have been essential in the molecular analysis of DDK-mediated regulation of MCM2-7 activation and the initiation phase of DNA replication. Here, the inhibitory activity of two distinct DDK inhibitor chemotypes, PHA-767491 and XL-413, were assessed in cell-free and cell-based proliferation assays. PHA-767491 and XL-413 show distinct effects at the level of cellular proliferation, initiation of DNA replication and replisome activity. XL-413 and PHA-767491 both reduce DDK-specific phosphorylation of MCM2 but show differential potency in prevention of S-phase entry. DNA combing and DNA replication assays show that PHA-767491 is a potent inhibitor of the initiation phase of DNA replication but XL413 has weak activity. Importantly, PHA-767491 decreased E2F-mediated transcription of the G1/S regulators cyclin A2, cyclin E1 and cyclin E2, and this effect was independent of CDK9 inhibition. Significantly, the enhanced inhibitory profile of PHA-767491 is mediated by potent inhibition of both DDK and the CDK2-Rb-E2F transcriptional network, that provides the molecular basis for its increased anti-proliferative effects in RB+ cancer cell lines.

Dbf4-Dependent Kinase: DDK-ated to post-initiation events in DNA replication.

Dbf4-Dependent Kinase (DDK) has a well-established essential role at origins of DNA replication, where it phosphorylates and activates the replicative MCM helicase. It also acts in the response to mutagens and in DNA repair as well as in key steps during meiosis. Recent studies have indicated that, in addition to the MCM helicase, DDK phosphorylates several substrates during the elongation stage of DNA replication or upon replication stress. However, these activities of DDK are not essential for viability. Dbf4-Dependent Kinase is also emerging as a key factor in the regulation of genome-wide origin firing and in replication-coupled chromatin assembly. In this review, we summarize recent progress in our understanding of the diverse roles of DDK.

Dysfunctional CAF-I reveals its role in cell cycle progression and differential regulation of gene silencing.

Chromatin Assembly Factor I (CAF-I) plays a central role in the reassembly of H3/H4 histones during DNA replication. In S. cerevisiae CAF-I is not essential and its loss is associated with reduced gene silencing at telomeres and increased sensitivity to DNA damage. Two kinases, Cyclin Dependent Kinase (CDK) and Dbf4-Dependent Kinase (DDK), are known to phosphorylate the Cac1p subunit of CAF-I, but their role in the regulation of CAF-I activity is not well understood. In this study we systematically mutated the phosphorylation target sites of these kinases. We show that concomitant mutations of the CDK and DDK target sites of Cac1p lead to growth retardation and significant cell cycle defects, altered cell morphology and increased sensitivity to DNA damage. Surprisingly, some mutations also produced flocculation, a phenotype that is lost in most laboratory strains, and displayed elevated expression of FLO genes. None of these effects is observed upon the destruction of CAF-I. In contrast, the mutations that caused flocculation did not affect gene silencing at the mating type and subtelomeric loci. We conclude that dysfunctional CAF-I produces severe phenotypes, which reveal a possible role of CAF-I in the coordination of DNA replication, chromatin reassembly and cell cycle progression. Our study highlights the role of phosphorylation of Cac1p by CDK and a putative role for DDK in the transmission and re-assembly of chromatin during DNA replication.

Roles of CDK and DDK in Genome Duplication and Maintenance: Meiotic Singularities.

Cells reproduce using two types of divisions: mitosis, which generates two daughter cells each with the same genomic content as the mother cell, and meiosis, which reduces the number of chromosomes of the parent cell by half and gives rise to four gametes. The mechanisms that promote the proper progression of the mitotic and meiotic cycles are highly conserved and controlled. They require the activities of two types of serine-threonine kinases, the cyclin-dependent kinases (CDKs) and the Dbf4-dependent kinase (DDK). CDK and DDK are essential for genome duplication and maintenance in both mitotic and meiotic divisions. In this review, we aim to highlight how these kinases cooperate to orchestrate diverse processes during cellular reproduction, focusing on meiosis-specific adaptions of their regulation and functions in DNA metabolism.

RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells.

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells.

CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor I with chromatin.

Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28-1 mutant and to a lesser extent in a cdc7-1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.

Behavior of replication origins in Eukaryota - spatio-temporal dynamics of licensing and firing.

Although every organism shares some common features of replication, this process varies greatly among eukaryotic species. Current data show that mathematical models of the organization of origins based on possibility theory may be applied (and remain accurate) in every model organism i.e. from yeast to humans. The major differences lie within the dynamics of origin firing and the regulation mechanisms that have evolved to meet new challenges throughout the evolution of the organism. This article elaborates on the relations between chromatin structure, organization of origins, their firing times and the impact that these features can have on genome stability, showing both differences and parallels inside the eukaryotic domain.

Increased Cdc7 expression is a marker of oral squamous cell carcinoma and overexpression of Cdc7 contributes to the resistance to DNA-damaging agents.

Cdc7-Dbf4 kinase (Dbf4-dependent kinase, DDK) is an essential factor of DNA replication and DNA damage response (DDR), which is associated with tumorigenesis. However, Cdc7 expression has never been associated to the outcome of oral squamous cell carcinoma (OSCC) patients, and the mechanism underlying cancer cell survival mediated by Cdc7 remains unclear. The Cdc7 protein expression of 105 OSCC tumor and 30 benign tissues was examined by immunohistochemistry assay. Overall survival rates of 80 OSCC patients were measured using Kaplan-Meier estimates and the log-rank tests. Cdc7 overexpression by adenovirus system was used to scrutinize the underlying mechanism contributed to cancer cell survival upon DDR. In silico analysis showed that increased Cdc7 is a common feature of cancer. Cdc7 overexpression was found in 96 of 105 (91.4%) studied cases of OSCC patients. Patients with higher Cdc7 expression, either categorized into two groups: Cdc7 high expression (2+ to 3+) versus Cdc7 low expression (0 to 1+) [hazard ratios (HR)=2.6; 95% confidence interval (CI)=1.28-5.43; P=0.0087] or four groups (0 to 3+) [HR=1.71; 95% CI=1.20-2.44; P=0.0032], exhibited a poorer outcome. Multivariate analysis showed that Cdc7 is an independent marker for survival prediction. Overexpressed Cdc7 inhibits genotoxin-induced apoptosis to increase the survival of cancer cells. In summary, Cdc7 expression, which is universally upregulated in cancer, is an independent prognostic marker of OSCC. Cdc7 inhibits genotoxin-induced apoptosis and increases survival in cancer cells upon DDR, suggesting that high expression of Cdc7 enhances the resistance to chemotherapy.