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2009 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Figure 2 Figure 2. Functional regions of Dbf4 determined by deletion analysis. Dbf4 contains three regions called motifs N, M, and C that are conserved among Dbf4 orthologs. A BRCT-like domain spans motif N. Motif C encodes a single C 2 H 2 type Zn-finger. Finally, two regions spanning motif M and C interact with Cdc7 and activate its kinase activity. In Figure 2, we summarize our analysis of Dbf4 functional regions. The N-terminal third of Dbf4 is dispensable for DNA replication. However, the N-terminus encodes several functions that are important for Dbf4 function. There are two putative classic nuclear localization sequences (NLSs) at 55-61 and 251-257. Although the first sequence is dispensable, deletion of both sequences is lethal. Functionality is restored to a dbf4 mutant lacking the first 265 residues by addition of a heterologous NLS from the SV40 large T-antigen, suggesting that there are no NLS sequences in the Dbf4 C-terminus. We also identified a BRCT-like domain from residues 115-219 that is apparently conserved in all Dbf4 orthologs. BRCT domains are often present in DNA damage-responsive proteins and they interact with phosphorylated residues. Although Dbf4 mutants deleting this region are viable, they exhibit a slow S-phase and defects in response to DNA-damaging agents such as hydroxyurea, bleomycin, and methylmethane sulfonate. Whether the BRCT-like region governs a DNA repair function for Dbf4 is uncertain, because addition of an SV40 NLS to the dbf4-ND221 deletion mutant reverses most of these DNA replication and damage phenotypes. This suggests that the damage sensitivity is a secondary consequence of lowered nuclear localization and, therefore, compromised initiation activity. Consistent with this explanation, we found that many initiation mutants also exhibit secondary DNA damage sensitivities. Interestingly, deletion of the BRCT-like domain causes defects in late-origin activation, but early origins are activated normally. This raises the intriguing possibility that the BRCT-like domain targets the kinase to late replication origins. We also constructed a series of C-terminal deletion Dbf4 mutants; such mutants that remove a conserved Zn-finger motif are viable. This indicates that C-terminal residues are not essential for Dbf4 activity. However, deletion of C-terminal residues results in a markedly slower S-phase progression, temperature sensitivity, and DNA damage sensitivity. This suggests that the C-terminus is required to activate full Cdc7 kinase activity or to target it to important replication substrates. Using recombinant Cdc7 and Dbf4 proteins, we found that Dbf4 mutants lacking the C-terminus have a profound defect in Cdc7 kinase activation. The Zn-finger motif also interacts with Cdc7 via a two-hybrid assay, and this interaction depends on conserved residues in the Zn-finger. Lastly, there is a second Cdc7 binding site that overlaps motif M. We found that either Cdc7 binding region could be deleted individually and still allow Cdc7 binding, but deletion of both domains does not allow Cdc7 binding. We would like to identify proteins that interact with the Dbf4 N-terminus and determine the functional consequences of those interactions. Clearly the N-terminal third of Dbf4 is not required for DNA replication, but these residues are conserved in mouse and human cells and so must confer some critical function. Using a two-hybrid approach, we found that the Dbf4 N-terminus interacts with Polo kinase, a key regulator of mitotic progression. Detailed analysis of this interaction suggests that Dbf4 influences chromosome segregation, which represents a totally new activity for Cdc7-Dbf4 kinase. 66

VARI | 2009 External Collaborators Catherine Fox, University of Wisconsin–Madison Carol Newlon, University of Medicine and Dentistry of New Jersey, Newark Philippe Pasero, CNRS, Montpellier, France Alain Verreault, University of Montreal, Quebec, Canada Wolfgang Zachariae, Max Plank Institute, Dresden, Germany Recent Publications Harkins, V., Carrie Gabrielse, L. Haste, and M. Weinreich. In press. Budding yeast Dbf4 sequences required for Cdc7 kinase activation and identification of a functional relationship between the Dbf4 and Rev1 BRCT domains. Genetics. Miller, Charles T., Carrie Gabrielse, Ying-Chou Chen, and Michael Weinreich. 2009. Cdc7p-Dbf4p regulates mitotic exit by inhibiting polo kinase. PLoS Genetics 5(5): e1000498. Bonte, Dorine, Charlotta Lindvall, Hongyu Liu, Karl Dykema, Kyle Furge, and Michael Weinreich. 2008. Cdc7-Dbf4 kinase overexpression in multiple cancers and tumor cell lines is correlated with p53 inactivation. Neoplasia 10(9): 920–931. Chang, FuJung, James F. Theis, Jeremy Miller, Conrad A. Nieduszynski, Carol S. Newlon, and Michael Weinreich. 2008. Analysis of chromosome III replicators reveals an unusual structure for the ARS318 silencer origin and a conserved WTW sequence within the origin recognition complex binding site. Molecular and Cellular Biology 28(16): 5071–5081. Fox, Catherine A., and Michael Weinreich. 2008. Beyond heterochromatin: SIR2 inhibits the initiation of DNA replication. Cell Cycle 7(21): 3330–3334. From left: Savreux, Gabrielse, Chang, Miller, Chen, Weinreich 67

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