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Journal of Biomolecular Screening (1)
The Journal of Cell Biology (1)
Greenberg, Roger A. (2)
Messick, Troy E. (2)
Amelina, Aljona (1)
Coleman, Kara A. (1)
Erkko, Hannele (1)
Karppinen, Sanna-Maria (1)
Lieberman, Paul M. (1)
Messick, Troy (1)
Morrissey, Devon (1)
Nikkilä, Jenni (1)
Pylkäs, Katri (1)
Reichman, Melvin (1)
Schultz, David C. (1)
Thompson, Scott (1)
Winqvist, Robert (1)
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Development of a High-Throughput Screen for Inhibitors of Epstein-Barr Virus EBNA1
Schultz, David C.
Lieberman, Paul M.
Journal of Biomolecular Screening
Latent infection with Epstein-Barr Virus (EBV) is a carcinogenic cofactor in several lymphoid and epithelial cell malignancies. At present, there are no small molecule inhibitors that specifically target EBV latent infection or latency-associated oncoproteins. EBNA1 is an EBV-encoded sequence-specific DNA-binding protein that is consistently expressed in EBV-associated tumors and required for stable maintenance of the viral genome in proliferating cells. EBNA1 is also thought to provide cell survival function in latently infected cells. In this work we describe the development of a biochemical high-throughput screening (HTS) method using a homogenous fluorescence polarization (FP) assay monitoring EBNA1 binding to its cognate DNA binding site. An FP-based counterscreen was developed using another EBV-encoded DNA binding protein, Zta, and its cognate DNA binding site. We demonstrate that EBNA1 binding to a fluorescent labeled DNA probe provides a robust assay with a Z-factor consistently greater than 0.6. A pilot screen of a small molecule library of ~14,000 compounds identified 3 structurally related molecules that selectively inhibit EBNA1, but not Zta. All three compounds had activity in a cell-based assay specific for the disruption of EBNA1 transcription repression function. One of the compounds was effective in reducing EBV genome copy number in Raji Burkitt lymphoma cells. These experiments provide a proof-of-concept that small molecule inhibitors of EBNA1 can be identified by biochemical high-throughput screening of compound libraries. Further screening in conjunction with medicinal chemistry optimization may provide a selective inhibitor of EBNA1 and EBV latent infection.
The ubiquitin landscape at DNA double-strand breaks
Greenberg, Roger A.
The Journal of Cell Biology
The intimate relationship between DNA double-strand break (DSB) repair and cancer susceptibility has sparked profound interest in how transactions on DNA and chromatin surrounding DNA damage influence genome integrity. Recent evidence implicates a substantial commitment of the cellular DNA damage response machinery to the synthesis, recognition, and hydrolysis of ubiquitin chains at DNA damage sites. In this review, we propose that, in order to accommodate parallel processes involved in DSB repair and checkpoint signaling, DSB-associated ubiquitin structures must be nonuniform, using different linkages for distinct functional outputs. We highlight recent advances in the study of nondegradative ubiquitin signaling at DSBs, and discuss how recognition of different ubiquitin structures may influence DNA damage responses.
Familial breast cancer screening reveals an alteration in the RAP80 UIM domain that impairs DNA damage response function
Coleman, Kara A.
Greenberg, Roger A.
Germline mutations in two major susceptibility genes, BRCA1 and BRCA2, account for nearly 20% of familial breast cancers. A majority of the remaining genetic factors involved in heritable breast cancer susceptibility are, however, unknown. Recently, a new BRCA1-interacting protein, RAP80, was identified. RAP80 plays an important role in BRCA1-mediated DNA damage responses by recruiting BRCA1 to DNA double-strand breaks. A comprehensive screening of DNA from affected index cases of 112 BRCA1/BRCA2 mutation-negative Finnish breast cancer families revealed altogether 10 alterations in RAP80, one of which, c.241-243delGAA, resulted in a single glutamic acid deletion at residue 81 in a highly conserved region of ubiquitin interaction motif 1. The resultant delE81 protein product displayed significantly reduced ubiquitin binding and double-strand break localization. Expression of the RAP80 delE81 allele impaired both BRCA1 and ABRA1 double-strand break recruitment, thus compromising BRCA1-mediated DNA damage response signaling. Compared to wildtype RAP80, expression of the delE81 allele was associated with a significant increase in cytogenetically detectable chromosomal aberrations, particularly chromatid breaks. Although evidently quite rare, these results suggest that critical constitutional mutations in RAP80 abrogate DNA damage response function and may be involved in genetic predisposition to cancer.
RAP80; breast cancer; BRCA1; ubiquitin
Results 1-3 (3)
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