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DNA Replication

Christian Speck

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Medical Research Council (MRC)
The precise duplication of chromosomal DNA is essential to preserve the genetic complement of the cell. In multicellular organisms mistakes during genetic inheritance can lead to a cell population that proliferates uncontrolled. To ensure that chromosomes only replicate once per cell cycle the process of chromosome duplication is divided into discrete steps which have to happen in a specific order for successful assembly of a DNA replication machine. The first major step is licensing of chromosomes during late in mitosis or early G1 phase. This reaction involves i.) Binding of the six-subunit Origin Recognition Complex (ORC) to origin DNA and ii.) Cdc6 and Cdt1 dependent loading of Mini-Chromosome maintenance proteins (MCM), a putative DNA helicase, onto chromatin to form the pre-replicative complex (pre-RC). Once the cell is committed to cell division and DNA replication, Dbf4 Dependent Kinase (DDK) and Cyclin Dependent Kinases (CDKs) get activated. In the second major step several proteins are recruited to origins, including Replication Protein A (RPA) and DNA polymerases, which lead to pre-initiation complex (pre-IC) formation and DNA replication. Origins that replicated get inactivated by S-phase specific cyclins to restrict DNA replication to once per cell cycle.||We are interested in understanding the function, mechanism and regulation of the multiprotein machine that assembles in a multistep process on DNA resulting in duplication of the genome. To address these questions we use yeast as a model system, since it is the only biochemical accessible system available that allow sequence specific complex assembly and will serve as good basis for a general understanding of eukaryotic DNA replication. The 1st aim is to determine the basic mechanisms in assembly and regulation of the preRC. Recently we analyzed the role of ORC and Cdc6 in pre-RC formation. Now our focus will be on Cdt1 and MCM proteins in pre-RC formation. We will analyze the interactions between Cdt1-MCM2-7 and ORC/Cdc6 in solution and on DNA using biochemical assays. We are interested in the question how Cdt1-MCM2-7 modifies the previously studied ORC-Cdc6-DNA complex. To analyze the function of MCM proteins we do assemble the entire pre-RC and also understand the binding/loading of MCM proteins. In collaboration with Huilin Li (Brokkhaven National Laboratory, USA) we will determine the 3D structures of multiple complexes on DNA with ORC, Cdc6, Cdt1 and MCM proteins. Structural and biochemical data will be consequently integrated into a model describing the function and mechanism in pre-RC formation. This information will be used to understand the consequences of pre-RC misregulation in cancer.

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