Accurate duplication of genetic material and its faithful segregation to daughter nuclei is of paramount importance for cell survival. Origin Recognition Complex (ORC) is a six-subunit protein complex that serves as the landing pad for the assembly of a multiprotein pre-replicative complex at DNA replication origins. ORC and other human replication initiation proteins have been shown to be required for replication of Epstein Barr virus (EBV) that is associated with Burkitt's lymphoma. Other than their bonafide roles in DNA replication, ORC proteins are required for heterochromatin organization, centrosome copy number maintenance, accurate cell cycle progression and cell division. We have identified a novel ORC associated protein, ORCA/LRWD1 that maps to 7q22.1 in human cells, a region that is frequently amplified in esophageal squamous cell carcinoma or shows rearrangements and loss of heterozygosity in myelodysplasia and acute leukemia. ORCA colocalizes with ORCs to telomeres and centromeres, structures critical for maintenance of genome integrity. Depletion of ORCA from human cells results in reduced binding of ORC proteins to chromatin and subsequent accumulation of cells in G1 phase. However, the roles of ORCA in preRC assembly and in stabilizing ORC to chromatin remain to be elucidated. The long-term goal of my laboratory is to understand how DNA replication and chromatin organization are coordinated in mammalian cells. The objective of the present proposal is to determine the roles played by ORCA in replication initiation and if its cell cycle regulation ensures that replication occurs 'once-and-oly-once' per cell cycle. Our hypothesis, based on preliminary data, is that ORCA-ORC interaction is crucial for specific loading of ORC to chromatin and for preRC assembly in G1 and chromatin organization in post-G1 cells. To test this hypothesis, we propose the following specific aims: 1) Determine the role of ORCA in preRC assembly; 2) Determine how ORCA stabilizes/recruites ORC to chromatin; and 3) Identify the mechanism of cell cycle regulation of ORCA and its biological significance. Our approach is innovative because we will characterize a novel component of the replication initiation machinery. This proposal is innovative in its combination of molecular biology, biochemistry with state of the art live cell imaging. My expertise in both molecular biology/ biochemistry and live cell imaging is quite unique and essential to this project. Several replication initiation proteins including Mini chromosome maintenance proteins are being used widely for cancer diagnostic purposes and it would be critical to pinpoint the function of ORC and ORCA in cancer and how they regulate genome stability. This proposal is therefore highly significant not only in understanding the basics of how replication and cell cycle progression is coordinated but also significant for the field of cancer biology.
The long-term focus of my laboratory is to understand the roles played by ORC and ORC associated proteins including ORCA/LRWD1 in replication initiation and how preRC assembly is linked to chromatin organization and cell cycle progression. In the present proposal we aim to study the role of ORCA in preRC assembly, its association with origins and how ORC protects the degradation of ORCA. Since ORCA plays a key role in DNA replication, studying the molecular basis of this would have direct implications for biomedical research.
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