DNA sequence data for many human exomes and genomes are accumulating at an ever-increasing pace, yet an understanding of the effect of small genetic variations on protein function lags behind. We have developed a method to analyze the function of up to ~1 million variants of a protein using next generation DNA sequencing and protein display formats that link genotype to phenotype. Our work to date has used small protein domains (typically <100 amino acids) and functional assays in yeast or in vitro. However, advances in technology suggest that we should be able to extend our approach to much larger proteins and more complex assays, in particular assays in human cells. Our overall goal is to develop the technology to rapidly assess the function, in human cells, of all th variants of a large human protein that has multiple activities and interactions. This technology will be prototyped using the BRCAl protein - in which germ-line mutations result in a vastly increased risk of breast and ovarian cancer-and then extended to other proteins implicated in cancer risk. We will compare the results from our assays to the data on disease risk and progression for known variants in order to establish the utility of our high throughput approach. Our Driving Biomedical Project will employ a DNA repair assay in human cells to analyze the activity of BRCAl variants.
Our specific aims are: 1) To generate all possible single amino acid changes in the BRCAl protein and to assess the variants for their proficiency in DNA repair (using the whole protein) and in E3 ligase activity (using a 304 amino acid domain);2) To compare the quantitative fitness of the BRCA1 variants obtained in our assays with a database of disease alleles;3) To extend the approach to other human genes relevant to cancer and amenable to similar assays, such as BRCA2, BARD1 and CHEK2.

Public Health Relevance

Recent advances are making it possible to determine the complete DNA sequence of any individual rapidly and inexpensively. Yet the differences in sequence revealed are often uninterruptable, because we do not understand how they affect the functions of the encoded proteins. We propose to develop methodology to assay for function tens of thousands of mutant versions of the breast and ovarian cancer susceptibility gene BRCAl and to extend this approach to other human genes implicated in cancer.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Biotechnology Resource Grants (P41)
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Special Emphasis Panel (ZRG1-BCMB-P (40))
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Sheeley, Douglas
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University of Washington
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Conkar, Deniz; Culfa, Efraim; Odabasi, Ezgi et al. (2017) The centriolar satellite protein CCDC66 interacts with CEP290 and functions in cilium formation and trafficking. J Cell Sci 130:1450-1462
Widjaja, Christella E; Olvera, Jocelyn G; Metz, Patrick J et al. (2017) Proteasome activity regulates CD8+ T lymphocyte metabolism and fate specification. J Clin Invest 127:3609-3623
Hope, Elyse A; Amorosi, Clara J; Miller, Aaron W et al. (2017) Experimental Evolution Reveals Favored Adaptive Routes to Cell Aggregation in Yeast. Genetics 206:1153-1167
Callens, Céline; Coelho, Nelson C; Miller, Aaron W et al. (2017) A multiplex culture system for the long-term growth of fission yeast cells. Yeast 34:343-355
May, Damon H; Tamura, Kaipo; Noble, William S (2017) Param-Medic: A Tool for Improving MS/MS Database Search Yield by Optimizing Parameter Settings. J Proteome Res 16:1817-1824
Hanna 4th, Michael G; Block, Samuel; Frankel, E B et al. (2017) TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER-Golgi intermediate compartments. Proc Natl Acad Sci U S A 114:E7707-E7716
Subramanian, Kanagaraj; Rauniyar, Navin; Lavalleé-Adam, Mathieu et al. (2017) Quantitative Analysis of the Proteome Response to the Histone Deacetylase Inhibitor (HDACi) Vorinostat in Niemann-Pick Type C1 disease. Mol Cell Proteomics 16:1938-1957
Zimmerman, Sandra G; Merrihew, Gennifer E; MacCoss, Michael J et al. (2017) Proteomics Analysis Identifies Orthologs of Human Chitinase-Like Proteins as Inducers of Tube Morphogenesis Defects in Drosophila melanogaster. Genetics 206:973-984
Ma, Yuanhui; McClatchy, Daniel B; Barkallah, Salim et al. (2017) HILAQ: A Novel Strategy for Newly Synthesized Protein Quantification. J Proteome Res 16:2213-2220
Cao, Liwei; Diedrich, Jolene K; Kulp, Daniel W et al. (2017) Global site-specific N-glycosylation analysis of HIV envelope glycoprotein. Nat Commun 8:14954

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