DNA replication is a fundamental process for all organisms to precisely duplicate genetic material prior to cell division. Central to the process is a helicase enzyme that uses ATP-hydrolysis to separate base-paired DNA to allow polymerases to gain access to synthesize complementary strands and also to drive the replication machinery along the DNA. In human and other eukaryotic cells, the helicase engine is the 6- protein MCM complex. The proposed research will fill a knowledge gap by providing atomic level mechanism pictures of MCM proteins as they interact with ATP compounds and with DNA. These will be studied at the molecular level by a coordinated approach involving structural studies by X-ray crystallography and cryo-electron microscopy and also in vitro methods to study their functions and interactions. The DEAD-box RNA helicase DDX3X has been implicated in many aspects of RNA processing and is associated with several human diseases, including HIV, HCV, and medulloblastoma. The proposed research will fill a knowledge gap by providing detailed pictures of DDX3X proteins interacting with RNA and ATP compounds in the functional state. A considerable body of preliminary data has been obtained for this project that includes the identification of a minimal fragment of DDX3X that has RNA- stimulated ATPase activity and its crystal structure bound to relevant substrates.
STATEMENT A major focus of the project is DNA replication, a fundamental event that is required in all life forms, and its disruption can lead to several forms of disease, including cancer. The central engine of the replication machinery is the hexameric MCM complex, which represents a logical, promising target for anti-cancer therapies. A second focus of the project is the human RNA helicase DDX3X, which is associated with several human diseases, including HIV, HCV, and the brain cancer medulloblastoma.
