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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Reddy, Michael K
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University of Illinois Urbana-Champaign
Anatomy/Cell Biology
Schools of Arts and Sciences
United States
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Anantharaman, Aparna; Gholamalamdari, Omid; Khan, Abid et al. (2017) RNA-editing enzymes ADAR1 and ADAR2 coordinately regulate the editing and expression of Ctn RNA. FEBS Lett 591:2890-2904
Zong, Xinying; Nakagawa, Shinichi; Freier, Susan M et al. (2016) Natural antisense RNA promotes 3' end processing and maturation of MALAT1 lncRNA. Nucleic Acids Res 44:2898-908
Jadaliha, Mahdieh; Zong, Xinying; Malakar, Pushkar et al. (2016) Functional and prognostic significance of long non-coding RNA MALAT1 as a metastasis driver in ER negative lymph node negative breast cancer. Oncotarget 7:40418-40436
Giri, Sumanprava; Chakraborty, Arindam; Sathyan, Kizhakke M et al. (2016) Orc5 induces large-scale chromatin decondensation in a GCN5-dependent manner. J Cell Sci 129:417-29
Anantharaman, Aparna; Jadaliha, Mahdieh; Tripathi, Vidisha et al. (2016) Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA. Sci Rep 6:34043
Giri, Sumanprava; Aggarwal, Vasudha; Pontis, Julien et al. (2015) The preRC protein ORCA organizes heterochromatin by assembling histone H3 lysine 9 methyltransferases on chromatin. Elife 4:
Kara, Nihan; Hossain, Manzar; Prasanth, Supriya G et al. (2015) Orc1 Binding to Mitotic Chromosomes Precedes Spatial Patterning during G1 Phase and Assembly of the Origin Recognition Complex in Human Cells. J Biol Chem 290:12355-69
Giri, Sumanprava; Prasanth, Supriya G (2015) Association of ORCA/LRWD1 with repressive histone methyl transferases mediates heterochromatin organization. Nucleus 6:435-41
Khan, Abid; Prasanth, Supriya G (2015) BEND3 mediates transcriptional repression and heterochromatin organization. Transcription 6:102-5
Khan, Abid; Giri, Sumanprava; Wang, Yating et al. (2015) BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase. Proc Natl Acad Sci U S A 112:8338-43

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