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2015 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report 2015 Michael Weinreich, Ph.D. Laboratory of Genome Integrity and Tumorigenesis Dr. Weinreich received his Ph.D. in biochemistry from the University of Wisconsin–Madison. He did a postdoctoral fellowship at Cold Spring Harbor Laboratory, New York, before joining VARI as a Scientific Investigator in March 2000. He is currently an Associate Professor. From left: Chang, Sasi, Minard, Weinreich Staff FuJung Chang, M.S. Michelle Minard Nanda Kumar Sasi, B.S. Students Nick Beam Clare Laut 58

Weinreich Research Interests We are studying several proteins that initiate DNA replication to learn how cells accurately maintain their genetic information. Cancer is caused by genetic and epigenetic mutations in DNA, and early mutations that impair cellular surveillance mechanisms can promote an increased mutation rate. The cumulative mutational burden can lead to the development of cancer as cells escape the normal growth, immunological, and proliferation controls. In addition, many of the proteins required for the initiation of DNA replication also have important roles in DNA repair pathways and other cell-cycle transitions. The Dbf4-dependent kinase (DDK; also known as Cdc7-Dbf4 kinase) is needed to initiate DNA replication at independent origins throughout the genome. It accomplishes this by phosphorylating and activating the MCM helicase, previously loaded in an inactive form at all origins during the G1 phase. DDK is also required for the accurate segregation of chromosomes during stress via a poorly understood mechanism. We recently reported that Dbf4 interacts with the Polo-like kinase Cdc5 to maintain the spindle position checkpoint. In yeast, Cdc5 facilitates chromosome separation during metaphase, entry into anaphase, spindle elongation, the exit from mitosis, and cytokinesis. We found that DDK binds to and inhibits Cdc5 when the mitotic spindle apparatus is not properly aligned between mother and daughter cells. Loss of this regulation can cause a significant increase in chromosome segregation errors and cell death; if the mitotic process were to continue with this misalignment, it would produce one multinucleate cell and one anucleate cell. However, the mechanism and regulation of Cdc5 by DDK is not well understood. Polo kinases such as Cdc5 contain two domains, a C-terminal polobox domain (PBD) that targets the kinase to its substrates and an N-terminal kinase domain. The PBD recognizes phosphorylated serine or threonine residues within a conserved S-pS/ pT-P/X motif. Although we previously defined a short peptide on Dbf4 that binds to the PBD, the PBD surface that interacts with Dbf4 was not known. We therefore used saturation mutagenesis to define residues within the PBD that are key to binding Dbf4, and in a second round of mutagenesis, we mapped a well-defined surface on the PBD—distinct from its pS/pT binding pocket—that binds to Dbf4. We can now investigate conserved interactions in human cells that might affect chromosome replication or segregation. Human cells contain four Polo-like kinases (Plks), several of which have increased expression in tumor cells. Dbf4 has evolved a checkpoint effector role to prevent chromosome segregation defects through binding and inhibiting Polo kinase. Our studies detailing the mechanism of Cdc5 binding to Dbf4 have defined a fundamentally new type of interaction, one perhaps used by other Cdc5 binding proteins. Many types of tumors show increased levels of both DDK and the Polo-like kinase Plk1 which, when inhibited, cause the death of many tumor cell types but not of normal cells. Because the ability of DDK to control multiple aspects of chromosome metabolism is likely conserved, it is crucial to understand these pathways for further development of highly effective chemotherapeutic agents and interventions. Recent Publications Corbi, Daniel, Sham Sunder, Michael Weinreich, Aikaterini Skokotas, Erica S. Johnson, and Edward Winter. 2014. Multisite phosphorylation of the Sum1 transcriptional repressor by S-phase kinases controls exit from meiotic prophase in yeast. Molecular and Cellular Biology 34(12): 2249–2263. Hiraga, Shin-ichiro, Gina M. Alvino, FuJung Chang, Hui-yong Lian, Akila Sridhar, Takashi Kubota, Bonita J. Brewer, Michael Weinreich, M.K. Raghuraman, et al. 2014. Rif1 controls DNA replication by directing protein phosphatase 1 to reverse Cdc7- mediated phosphorylation of the MCM complex. Genes and Development 28(4): 372–383. 59

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