Initiation of the eukaryotic chromosome replication is a tightly regulated multi-step process. Failure to ensure once and once only initiation event per cell division cycle can result in uncontrolled cell proliferation and tumorigenesis. Central to the replication initiation is the origin recognition complex (ORC) that recognizes the chromosomal origins of replication. With the help of replication initiator Cdc6 and replication licensing facto Cdt1, ORC recruits and loads the Mcm2-7 helicase onto DNA, forming a highly stable Mcm2-7 double hexamer. This completes the first milestone - formation of pre-replication complex (pre-RC). A series of subsequent events culminate to formation of pre-initiation complex (pre-IC) and leading cell to enter into the S phase. X-ray crystal structures have been reported for key proteins involved in prokaryotic replication initiation, but little is known about the structures o the corresponding eukaryotic complexes. This is so because the eukaryotic replication initiators assemble into large and transient complexes, which have been exceedingly difficult for crystallography. Recent advance in hardware and software has made cryo-EM an ideal approach to studying these complexes at much improved resolution. We therefore propose to study how the pre- Replication Complex is established by cryo-EM. This work will advance our knowledge in the initiation of eukaryotic chromosome replication.
The goal of this project is to study the structure and mechanism of protein complexes that initiate and facilitate DNA replication in eukaryotes. This work will fill the current gap in our knowledge in structures and interaction of the several important replication initiators. Yeast and human are two model systems for learning how DNA replication may occur in a cell. It has been well known that DNA and its associated chromatin structures must be duplicated in a tightly controlled and accurate manner, in order to maintain the integrity of the cellular genome from generation to generation. Interruption of these controls can destabilize genome and lead to tumorigenesis. Understanding the structure and interaction of essential DNA replication proteins will enable small molecule based therapeutic intervention of cancers and many other proliferative disorders.
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