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          Cell division cycle-7 kinase (Cdc7) in complex with Dbf4 and nucleotide (PDB ID: 4F99) is a serine/threonine kinase in humans that is essential for the initiation of eukaryotic DNA replication (1). Originally identified in budding yeast and later found in numerous eukaryotic homologs, the kinase function of Cdc7 has been evolutionarily conserved, suggesting that its role in DNA replication initiation is universal (2,3). Reports indicate that Cdc7 is required for activation of each origin on the chromosome during DNA synthesis. Cdc7 and Dbf4 are overexpressed in many cancers and tumor cell lines, and are a target for many cancer drug therapies (1). Additionally, there is evidence that Cdc7 plays roles in meiosis, maintenance of chromosome structures, and DNA repair (2).

DNA fragments encoding full length human Cdc7 or its deletion mutants were cloned between NcoI and XhoI sites of bacterial vector pRSFDuet1. Crystallization was successful with Cdc7 lacking noncanonical residues 1-36, 228-359, and 484-529 in complex with a fragment of Dbf4 containing only the conserved motifs M and C, residues 210-350 (referenced to as Cdc7(ΔN2q3b)–MC). This deletion construct maintained intrinsic specificity as well as 40-50% of wildtype Cdc7-Dbf4 kinase activity, with reduction caused by deletion of Cdc7 residues 228-359. Cdc7(ΔN2q3b)–MC) crystals were grown at 291K in hanging drops by vapor diffusion against a reservoir of 22-26% PEG-1500 and 15% glycerol. Structures were refined using diffraction collected from crystals grown in the presence of MgCl₂ and AMP-PNP.  The structure was solved by automatic molecular replacement using BALBES. Presence of a zinc atom bound to Dbf4 was verified by anomalous X-ray scattering and the nucleotide was modelled by ADP (1).

The product protein has an isoelectric point of 8.84 and molecular weight of 57458.24 Da, which was determined using the ExPASy database (4). It consists of two subunits: Cdc7 kinase (subunit A) and its activator, Dbf4 (subunit B) (2). The secondary structure of Cdc7 contains 35% α-helices 15% β-sheets and that of Dbf4 contains 15% α-helices 10% β-sheets. 3/10 helices and random coils are present to both to uncertain degrees (5). Three ligands are present in the protein complex: Zn²⁺, Mg²⁺ and a nucleotide (ADP), each essential to the structure and functionality of the active kinase (1).

Protein kinases are a large family of enzymes that catalyze the covalent transfer of an inorganic phosphate from Mg-ATP to Ser/Thr and Tyr side chains of other proteins. Though each is specific to its target and function, there are conserved structural aspects among kinases that are necessary for their activity, which Cdc7 possesses (1,6). Protein kinases contain an adenine pocket to which an Mg-ATP binds, and are present in this Cdc7 structure as Mg²⁺ and ADP ligands. ATP binding is necessary in vivo as it provides a phosphate group for the kinase to transfer to its target substrate. Though the necessity for Mg²⁺ is poorly understood, there is evidence that Mg²⁺ ions in the active sites could enhance the binding affinities of ATP to the adenine pocket (7). Cdc7 and other protein kinases also contain an active catalytic loop, where an aspartate residue necessary for carrying out kinase activity is located (1,6).

Cdc7 displays the bilobal tertiary architecture true to most protein kinases, consisting of an active site located in a deep cleft between an N- and C-terminal lobe (1,6).  The N-lobe of Cdc7 (residues 41-135) contains a five-stranded antiparallel β-sheet (β1-5), a single, well conserved α-helix (αC), and two additional helices at the N terminus (Nα1 and Nα2) (1). The C-lobe consists of mainly α-helices as well as two β-ribbons. It houses the activation segment, a sequence between conserved DFG (residues 196-198) and APE (residues 381-383) motifs. Within this sequence lies the catalytic loop, referring to the structural element that contains the catalytic aspartate residue required to phosphorylate the hydroxyl group of the target substrate (1,6). Bound to the active site region is a nucleotide (modelled by ADP) and Mg²⁺ ion. The nucleotide associates at an adenine pocket, formed by ionic interaction between adenine and residues Ala-88, Ile-64, Met-118, Met-134, Tyr-136, Leu-184, and Val-195. It is further bound by a hydrogen bond between its N1 adenine atom and the peptide amide of Leu-137. Additionally, Glu-104 projects from the αC helix to form a salt bridge with Lys90, which interacts with the phosphate groups of the nucleotide. The Mg²⁺ ion is coordinated by phosphate groups of the nucleotide and Cdc7 residues Asn-182 and Asp-196. The remainder of contact between the nucleotide and adenine-binding pocket of Cdc7 comprises of Van der Waals interactions (1).

Interaction with Dbf4 is necessary to activate the kinase activity of Cdc7. Cdc7 levels remain constant throughout the cell cycle, but its kinase activity is known to be stage-specific with maximal activity occurring at the G1/S transition (8). This fluctuation in kinase activity correlates with changes in levels of the regulatory subunit Dbf4 (9). Dbf4 wraps around Cdc7, burying approximately 3,000 Ų of each molecular surface through hydrophobic interactions, accounting for an estimated solvation energy gain of -41 kcal/mol. The regulatory subunit contains two structural motifs M and C which pack against Cdc7 C- and N- lobes, respectively.  Dbf4 motif M motif is composed of a pair of β-strands (β1 and β2) and a well-ordered coil region while motif C is composed of three α-helices (α1, α2 and α3) and a pair of β-strands (β3 and β4). The β1 strand of Dbf4 motif M forms an antiparallel β-sheet with β ribbons of the Cdc7 C-lobe. This association acts as a tethering domain that maintains effective association of the heterodimer complex. Motif C forms a Zn2+ binding domain, stabilized by Cys-296, Cys-299, His-309 and His-315. The Zn²⁺ binding domain stabilizes the αC of the Cdc7 N-terminal lobe, which is essential to induce the active kinase conformation of Cdc7. Disruption of this Zn²⁺-αC interaction greatly truncates kinase activity (1).

The major physiological target of Cdc7-Dbf4 kinase is minichromosomal maintenance protein (MCM), which plays a primary role in DNA replication as part of the pre-replication complex (3). In eukaryotes, DNA helicase is comprised of a heterohexamer of six MCM proteins (MCM2-7). The MCM2-7 complex is loaded at replication origins in an inactive form during G1-phase. As the cell enters S-phase, the helicase is activated in a phosphorylation process that requires both Cdc7 and cyclin dependent kinase (CDK), as well as recruitment of many other factors. Activation induces DNA melting, establishing two replication forks of opposite polarity (10).

The sequence of Cdc7(ΔN2q3b)–MC was analyzed through the Position-Specific Iterated Basic Local Assignment Search Tool (PSI-BLAST) to find proteins of the most similar primary structure. An E value is produced on the basis of sequence similarity, where a value less than 0.05 indicates high similarity. The sequence was additionally run through the Dali Server in order to find proteins of the most similar tertiary structure by calculating the differences in intramolecular distances. This produces a Z-score, where a Z-score over 2 indicates strong structural similarity.

Comparison with CDK2 in complex with FragLite37 (PDB: 6q4g) produces an E-value of 1x10^-23 and Z-score of 27.2, indicating great similarities in both primary and tertiary structures (11, 12). CDK is a cyclin dependent Ser/Thr kinase that is also necessary for the firing of replication origins. DNA replication initiation will not occur without kinase activity provided by both Cdc7 and CDK. Analysis of the structures of these two kinases is twofold: it reveals similarities due to conserved residues that pertain to kinase activity, as well as important differences that may elucidate the specific role each protein plays in DNA initiation. Just like Dbf4, the CDK2 structure contains canonical motifs RD, APE, and DFG. The locations, however, are different, lying at CDK2 residues 126-127, 170-172, and 145-147 respectively. Additionally, Cdc7 residues Lys-90 and Glu-104 that form an essential salt bridge in are matched in CDK2 by residues Lys-33 and Glu-51 (11, 13). Results from the Dali Server display extensive similarities in secondary structures as well as a CDK2 adopting a bilobal structure (12, 13). Superimposition of the two protein structures reveals highly related secondary and tertiary structures, which may account for similarities in target substrate (Appendix 1). A major difference between the structures lie in activator association where Cdc7 is activated by Dbf4 while CDK2 is activated by cyclin.

In conclusion, Dbf4 kinase activity is essential for the initiation of DNA replication at each origin. Its kinase activity is regulated by its activator, Dbf4, which associates with Cdc7 at two sites. Interaction between the C-terminal lobe of Cdc7 and Dbf4 M of Dbf4 provides tethering of the two proteins while interaction between the N-terminal lobe of Cdc7 and Dbf4 motif C induces the active kinase form of Cdc7. Truncated kinase function to Cdc7 can stop the cell cycle and proper growth, while overexpression may be a factor in formation of tumor cell lines. There is ample evidence behind the universality of eukaryotic DNA replication initiation, so it is important to further understand the mechanism behind Cdc7 function (1).