REVISED ABSTRACT Replication initiation in eukaryotes is believed to be dependent on a six subunit, ATP-dependent complex of proteins, the Origin Recognition Complex (ORC), which loads the helicase MCM2-7 at origins of replication. In the last cycle of this grant we made the surprising discovery that several human cancer cell lines continue to proliferate and replicate their DNA in the absence of two important subunits of ORC, ORC1 or ORC2. This proposal will test whether cancer cell-lines survive through the action of a crippled ORC (ORC missing one subunit), or because cell transformation activates an alternate helicase-loading mechanism that allows MCM2-7 loading in the absence of the six subunit ORC. The proposal will also identify how human ORC activates and represses the compactness of the chromatin and thus regulate gene expression. It will test whether the individual subunits of ORC have functions in this regard only as the complex ORC or as individual proteins independent of the holo-ORC. The results will delineate the importance of ORC in replication initiation and maintenance of genome stability in cancer cells, identify ORC-bypass mechanisms and identify replication-independent functions of ORC in regulating cell physiology.

Public Health Relevance

and Significance Regulation of genome stability is the hallmark of normal cells, and often deregulated in cancer cells. This project will determine whether the Origin Recognition Complex (ORC), till now believed to be essential for replication initiation, can be bypassed in cancer cells and the mechanism of this bypass. This will be very important for understanding how genome instability occurs in cancers. Non-homologous end-joining (NHEJ) mediated repair of double-strand breaks in DNA is important for the resistance of a cancer cell to radiotherapy and common types of chemotherapy. By implicating a novel family of protein kinases in the signal transduction from DSB to activation of NHEJ, the project will identify new targets for drugs that will increase the sensitivity of cancer cells to radiotherapy or chemotherapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA060499-25
Application #
9875379
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Fingerman, Ian M
Project Start
1994-07-11
Project End
2025-01-31
Budget Start
2020-02-01
Budget End
2021-01-31
Support Year
25
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Virginia
Department
Biochemistry
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Paulsen, Teressa; Kumar, Pankaj; Koseoglu, M Murat et al. (2018) Discoveries of Extrachromosomal Circles of DNA in Normal and Tumor Cells. Trends Genet 34:270-278
Abbas, Tarek; Dutta, Anindya (2017) Regulation of Mammalian DNA Replication via the Ubiquitin-Proteasome System. Adv Exp Med Biol 1042:421-454
Kumar, Pankaj; Dillon, Laura W; Shibata, Yoshiyuki et al. (2017) Normal and Cancerous Tissues Release Extrachromosomal Circular DNA (eccDNA) into the Circulation. Mol Cancer Res 15:1197-1205
Lee, Kyung Yong; Im, Jun-Sub; Shibata, Etsuko et al. (2017) ASF1a Promotes Non-homologous End Joining Repair by Facilitating Phosphorylation of MDC1 by ATM at Double-Strand Breaks. Mol Cell 68:61-75.e5
Reon, Brian J; Dutta, Anindya (2016) Biological Processes Discovered by High-Throughput Sequencing. Am J Pathol 186:722-32
Shibata, Etsuko; Kiran, Manjari; Shibata, Yoshiyuki et al. (2016) Two subunits of human ORC are dispensable for DNA replication and proliferation. Elife 5:
Dillon, Laura W; Kumar, Pankaj; Shibata, Yoshiyuki et al. (2015) Production of Extrachromosomal MicroDNAs Is Linked to Mismatch Repair Pathways and Transcriptional Activity. Cell Rep 11:1749-59
Lee, Kyung Yong; Dutta, Anindya (2015) Initiation of replication in Xenopus egg extracts at a spatially defined origin. Cell Cycle 14:2391
Lee, Kyung Yong; Im, Jun-Sub; Shibata, Etsuko et al. (2015) MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex. Nat Commun 6:7744
Im, Jun-Sub; Keaton, Mignon; Lee, Kyung Yong et al. (2014) ATR checkpoint kinase and CRL1?TRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks. Genes Dev 28:875-87

Showing the most recent 10 out of 89 publications