DNA Replication

The duplication of the genome has to function in a precise regulated way with errors resulting potentially in promoting cancer and other diseases. A multi-protein machine, which performs the duplication process, assembles onto DNA every time the DNA is duplicated and consequently disassembles once the process is finished. We study the process of the assembly pathway, which is frequently misregulated in cancer. To analyze the DNA duplication mechanisms we are using yeast as a model system, since the major DNA duplication mechanism are conserved from yeast to human and yeast has genetically defined start sites on the DNA to initiate DNA duplication. We use biochemical and structural methods to understand the properties and shape of the proteins and the DNA involved. We are specifically interested in how the protein machine chooses the perfect spot on the DNA for assembly, how certain proteins function to load a ring shaped protein complex onto the rod shaped DNA and the consequences to the protein-DNA interface. Consequently we want to understand the function of the round shape protein complex on the DNA, which potentially promotes separation of the 2 strands of DNA during duplication. Also we would like to understand more about the regulation of the DNA duplication process, where some regulators are known to be misregulated in cancer. Therefore we are interested how regulators influence key processes during assembly, which we monitor to address their role.

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.