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"THE NF90/NF45 COMPLEX AND GENOME STABILITY"

by
Raghavendra A. Shamanna
Biochemistry and Molecular Biology Program
B.S. 2000, University of Agricultural Sciences, Bangalore, India
M.S. 2002, University of Agricultural Sciences, Dharwad, India


Thesis Advisor: Michael B. Mathews, Ph.D.
Professor and Chair
Department of Biochemistry and Molecular Biology

Monday, February 20, 2012
12:00 P.M., MSB E-609b


Abstract

Nuclear factor 90 (NF90) is an essential human RNA-binding protein that forms a heterodimeric complex with nuclear factor 45 (NF45). Depletion of either protein results in the appearance of giant multinucleated cells.
To elucidate the mechanism underlying this phenotype, a cell line was constructed allowing inducible NF45 knockdown. Time-lapse microscopy revealed that binucleated cells arise by incomplete abscission of daughter cells followed by fusion. Multinucleate cells arise through aberrant division of binucleated cells, with features suggestive of DNA repair defects. Immunostaining demonstrated greatly increased numbers of chromosomal foci indicative of the presence of DNA double-strand breaks (DSB), and NF90- or NF45-depleted stable cell lines displayed decreased clonogenic survival after g-irradiation. NF90/NF45 had previously been reported to co-purify with the DNA-dependent protein kinase (DNA-PK), which participates in DSB repair via nonhomologous end joining (NHEJ). Co-immunoprecipitation experiments confirmed this interaction. We therefore hypothesized that NF90/NF45 modulates the function of DNA-PK in NHEJ.
NF90-specific antibody was found to inhibit DNA end-joining in vitro, and NF90/NF45 immunodepletion impaired NHEJ in a novel assay system. Correspondingly, NF90/NF45 knockdown reduced NHEJ activity in vivo. Thus, we conclude that the NF90/NF45 complex regulates DNA damage repair via DNA-PK, and that dysregulation of the repair system leads to the multinucleated phenotype. Furthermore, removal of RNA from the in vitro NHEJ assay system reduced DNA end-joining activity, implying that NHEJ may be modulated by RNA bound to the NF90/NF45 complex.
This complex also regulates gene expression. NF90/NF45 knockdown increased the levels of p21 and p53 in HeLa cells, which normally contain little p53 because of rapid degradation mediated by the human papilloma virus (HPV) E6 oncoprotein. Induction of p21 and p53 by NF90/NF45 depletion was restricted to HPV-derived cervical carcinoma cell lines and sensitized HeLa cells to apoptosis synergistically with the anticancer drug camptothecin. Molecular analysis indicated that NF90/NF45 depletion reduces transcription from the HPV early promoter; the resultant increase in p53 protein restores p21 transcription. Thus, NF90/NF45 is co-opted by HPV to activate viral oncogene expression.
These studies have uncovered two hitherto unknown functions of NF90/NF45, revealing its importance for the maintenance of genome stability in cells.


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