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2009 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Michael Weinreich, Ph.D. Laboratory of Chromosome Replication Dr. Weinreich received his Ph.D. in biochemistry from the University of Wisconsin–Madison in 1993. He then was a postdoctoral fellow in the laboratory of Bruce Stillman, Director of the Cold Spring Harbor Laboratory, New York, from 1993 to 2000. Dr. Weinreich joined VARI as a Scientific Investigator in March 2000. He was promoted to Senior Scientific Investigator in September 2008. Staff Dorine Savreux, Ph.D. FuJung Chang, M.S. Carrie Gabrielse, B.S. Students Ying-Chou Chen, M.S. Charles Miller, B.S. Christina Gourlay Caitlin May Christina Untersperger 64

VARI | 2009 Research Interests We study early events that promote the initiation of DNA synthesis, which occurs at specific sequences termed replication origins. Various genome-wide approaches have identified from 320 to 420 possible replication origins in budding yeast, and there are perhaps 10,000 origins in human cells. The initiation of DNA replication occurs in a temporally distinct manner during G1 and S phase, and no origin initiates replication (fires) more than once per cell cycle to maintain normal diploid content. In G1 phase, each origin assembles approximately 40 polypeptides in a temporally defined order, culminating in the initiation of DNA replication at the G1/S phase boundary. The first stage of this process is called pre-replicative complex assembly and requires the origin recognition complex (ORC), Cdc6, and Cdt1. ORC directly binds to origin sequences and then recruits Cdt1 and Cdc6 during G1 phase. These three proteins cooperate to load the MCM DNA helicase at origins in an ATP-dependent reaction. Cyclin-dependent kinases and the Cdc7-Dbf4 kinase then catalyze the association of additional proteins with the MCM helicase to activate it, ultimately causing unwinding of the duplex DNA and the initiation of bidirectional DNA synthesis (Figure 1). In our lab we study three key aspects of DNA replication: 1) Replication origin structure 2) How Cdc6-ATP functions to load the MCM helicase within a chromatin context 3) How Cdc7-Dbf4 kinase contributes to the normal cell cycle and human malignancies Figure 1 Studies to understand the basic molecular biology of DNA replication and cell cycle progression are highly relevant for cancer biology, given that malignant cells often contain mutations in the cell growth and checkpoint pathways that drive normal proliferation. Here we describe recent studies on the Cdc7-Dbf4 kinase, which is a crucial regulator of DNA replication in all eukaryotic cells. Cdc7-Dbf4 is a conserved, two-subunit, serine/threonine protein kinase that catalyzes DNA synthesis at individual replication origins. Cdc7-Dbf4 promotes DNA synthesis after MCM helicase loading at the origin, likely by activating its helicase activity. This leads to origin unwinding and the assembly of DNA polymerases that initiate bidirectional DNA synthesis. Although Cdc7 is a member of the protein kinase superfamily, it requires the Dbf4 regulatory subunit to activate its kinase activity. We have determined the regions of Dbf4 that bind to and activate Cdc7 kinase by mutational analysis, and we are also investigating how Dbf4 targets Cdc7 kinase to its various substrates in the cell. 65

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