High impact information on DBF4

• A screen based on two- and one-hybrid approaches demonstrates that the product of the DBF4 gene interacts with yeast replication origins in vivo [1].
• The analysis described here suggests a model in which one function of Dbf4 may be to recruit the Cdc7 protein kinase to initiation complexes [1].
• The association of Dbf4p with a detergent-insoluble chromatin fraction in G(1)-phase nuclei requires ORC but not Cdc6p or Clb/Cdc28 kinase activity, and correlates with competence for initiation [2].
• The dbf4 suppressor mutation restores competence to initiate DNA synthesis to the mcm2-1 mutant [3].
• The Dbf4p phosphorylation in response to HU is RAD53 dependent [4].

Biological context of DBF4

• CDC45 is required in conjunction with CDC7/DBF4 to trigger the initiation of DNA replication [5].
• The DBF4 allele dna52-1 was cloned and sequenced, revealing a single point mutation within the M motif [6].
• The gene was identified as DBF4, which was previously isolated by complementation in dbf4-1 mutant cells and is required for the G1----S phase progression of the cell cycle [7].
• These results suggest that the CDC7 and DBF4 polypeptides interact directly or indirectly to permit initiation of yeast chromosome replication [7].
• Conversely, CDC7 on a multicopy plasmid suppresses the dbf4-1, -2, -3 and -4 mutations but not dbf4-5 and DBF4 deletion mutations [7].

Anatomical context of DBF4

• Eukaryotic cells coordinate chromosome duplication by assembly of protein complexes at origins of DNA replication and by activation of cyclin-dependent kinase and Cdc7p-Dbf4p kinase [4].
• The Drosophila chiffon gene is required for chorion gene amplification, and is related to the yeast Dbf4 regulator of DNA replication and cell cycle [8].
• Furthermore, a fused polypeptide containing only Dbf4 motifs M and C without any spacer can activate Hsk1 and is capable of rescuing the growth defect of him1 null cells [9].

Associations of DBF4 with chemical compounds

• We used gamma-irradiation to induce DNA damage and hydroxyurea to limit deoxyribonucleotides in cells deprived of DBF4 function to investigate the requirement for DNA replication in DNA-responsive checkpoints [10].
• Deoxyribonucleoside triphosphate levels increased severalfold at G1/S in wild-type cells and in dbf4 mutants incubated at the non-permissive temperature [11].
• On the other hand, internal deletion of Dbf4 motif N, which has some similarity with the BRCA C-terminal domain motif, results in a defect in hydroxyurea-induced checkpoint responses and sensitivity to methyl methane sulfonate, yet mitotic functions and kinase activation are intact [9].
• Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the K(m) for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme [12].
• A generic time-resolved fluorescence assay for serine/threonine kinase activity: application to Cdc7/Dbf4 [12].

Physical interactions of DBF4

• An active Clb/Cdc28 kinase complex, or its vertebrate equivalent, is required in trans to stimulate initiation in G(1)-phase nuclei, whereas the Dbf4/Cdc7 kinase complex must be provided by the template nuclei themselves [2].
• We had previously shown that Dbf4p interacts with the M phase polo-like kinase Cdc5p, a key regulator of the APC late in mitosis [13].

Regulatory relationships of DBF4

• Temperature-sensitive cdc7 mutations of Saccharomyces cerevisiae are suppressed by the DBF4 gene, which is required for the G1/S cell cycle transition [7].
• These findings suggest that S-CDKs and Cdc7p-Dbf4p promote loading of Cdc45p and RPA onto a preformed prereplication complex at each origin with preprogrammed timing [14].

Other interactions of DBF4

• We propose a model in which Dbf4p targets Cdc7p to the prereplication complex prior to the G(1)/S transition, by a pathway parallel to, but independent of, the Cdc6p-dependent recruitment of MCMs [2].
• These data indicate that CDC45 functions in late G1 phase in concert with CDC7/DBF4 to trigger initiation at replication origins after the assembly of the prereplicative complexes [5].
• A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5 [15].
• CDC7/DBF4 functions in the translesion synthesis branch of the RAD6 epistasis group in Saccharomyces cerevisiae [16].
• Previously, a 186-amino-acid Dbf4 region that mediates interactions with both the origin recognition complex and Rad53 was identified [6].

Analytical, diagnostic and therapeutic context of DBF4

• Using fluorescence in situ hybridization (FISH), the human DBF4 gene was localized to chromosome 7 (q21.3), whereas FISH mapped the murine counterpart to band A2 on chromosome 5 [17].
• A previous Dbf4p multiple sequence alignment identified a conserved approximately 40-residue N-terminal region with similarity to the BRCA1 C-terminal (BRCT) motif called "motif N." BRCT motifs encode approximately 100-amino-acid domains involved in the DNA damage response [18].
• This is caused by titration of a regulatory protein, Dbf4, from the wild-type Cdc7, which leads to inactivation of its kinase activity [19].

References