2008 Scientific Report

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Van Andel Research Institute | Scientific Report Figure 2 Figure 2. Model for SIR2- mediated inhibition of DNA replication. A) Schematic of the ARS315 element that contains an inhibitory sequence element (I S ) downstream of the site for pre-RC assembly. B) The I S element positions a nucleosome close to B2, which is inhibitory for pre-RC assembly. Deletion of SIR2 or mutation of the I S element allows repositioning of the nucleosome to allow pre-RC assembly. Cdc7p-Dbf4p is a two-subunit protein kinase required for initiating DNA replication after MCM helicase loading. The Cdc7p kinase subunit binds Dbf4p, which activates its kinase activity. Although Cdc7p-Dbf4p is required to promote DNA replication, it also has an undefined role in the repair of certain DNA lesions. In order to define the amino acids required for its roles in DNA replication and repair, we are analyzing Dbf4p using a mutational approach. We found that the Dbf4p N-terminus is dispensable for DNA replication, but it encodes functions that participate in the repair of DNA lesions and the firing of late-replication origins. An N-terminal BRCT-like motif may mediate these activities. It is also possible that Dbf4p maintains replication fork stability by targeting Cdc7p kinase to stalled replication forks. Interestingly, these are separable activities from Dbf4p’s essential role in promoting initiation of DNA replication. In addition, we have identified two regions within Dbf4p—a C-terminal Zn-finger motif and a separate region—that mediate binding to and activation of the Cdc7p kinase. The C-terminal region is also not essential for Dbf4p activity, but loss of this region dramatically lowers Cdc7p kinase activity. We also are studying the human Cdc7-Dbf4 protein kinase. We previously raised monoclonal antibodies that recognize both human subunits and used these to screen human cancer cell lines and primary human tumors for HsCdc7-Dbf4 abundance. Although both subunits are expressed at very low levels in normal cycling cells (and are perhaps absent in post-mitotic cells), they are up-regulated in a substantial number of tumor cell lines. HsCdc7 protein is also highly expressed in some primary breast and colon tumors. By screening expression data from a panel of more than 650 primary human tumors, we also found that CDC7 and DBF4 mRNA expression are coordinately up-regulated in many tumors of diverse origin. Similarly, we screened several primary tumors and tumor cell lines for gene copy changes in CDC7 and DBF4. We found that the DBF4 gene copy number is often elevated in those cells expressing higher levels of Cdc7-Dbf4 kinase. Because HsCdc7 is an essential kinase for DNA replication, its increased expression level in some tumors and tumor cell lines may reflect higher rates of cellular proliferation. However, we find no correlation between doubling time and Cdc7-Dbf4 expression in the NCI60 tumor cell lines. Furthermore, several published studies suggest that increased Cdc7-Dbf4 expression can inhibit the growth of rodent cell lines but has no effect on the growth of human cells. We suggest that since HsCdc7-Dbf4 is likely involved in other aspects of chromosome metabolism (e.g., DNA repair) and functions in the S-phase checkpoint, its increased expression in some tumor cell lines may offer an advantage for handling the chromosome instability that occurs in many human tumors. Our aim is to understand the mechanism(s) that allow increased expression of Cdc7-Dbf4 kinase in tumor cells and to investigate its phenotypic effects. 70
VARI | 2008 From left: Savreux, Gabrielse, Chang, Miller, Chen, Weinreich External Collaborators Angelika Amon, Massachusetts Institute of Technology, Cambridge Catherine Fox, University of Wisconsin–Madison Carol Newlon, University of Medicine and Dentistry of New Jersey, Newark Alain Verreault, University of Montreal, Quebec, Canada Recent Publications Crampton, Amber, FuJung Chang, Donald L. Pappas, Jr., Ryan L. Frisch, and Michael Weinreich. 2008. An ARS element inhibits DNA replication through a SIR2-dependent mechanism. Molecular Cell 30(2): 156–166. 71
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2008 Scientific Report

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