We have two main research interests. First, we are interested in understanding how transcription complexes are assembled on promoters and how their activity is regulated. Our work focuses on the Snf2/Swi2-related protein Mot1. Mot1 is an essential protein that regulates transcription by a remarkable mechanism: it uses ATP hydrolysis to disrupt TATA-binding protein (TBP)-DNA complexes. Since TBP provides a platform for assembly of the preinitiation complex (PIC), Mot1 activity has many diverse effects on transcription genome-wide. Displacement of TBP from chromatin can prevent the formation of transcription complexes and thereby inhibit transcription. By functioning in TBP-DNA surveillance, Mot1 also prevents transcription complexes from assembling at nonpromoter regions and it participates in the timely deactivation of gene expression in response to environmental cues. Paradoxically, Mot1 also functions as a transcriptional activator of some genes, a still rather mysterious activity that we are very interested in understanding. We employ molecular, genetic, and genome-scale approaches using the yeast Saccharomyces cerevisiae to understand the dynamic behavior of PIC components in vivo and to define Mot1's role in regulating this dynamic behavior. Our focus on Mot1 is also of interest because it is a member of a large enzyme family with diverse roles in regulating chromatin metabolism. We are employing biochemical and biophysical approaches using purified components to better define Mot1's structural organization, the architecture of the complexes it forms with TBP and DNA, as well as Mot1's catalytic mechanism. As the Mot1 ATPase is highly related to other Snf1/Swi2 ATPases, our results are relevant for understanding more generally how enzymes in this class function.

A second area of interest is in understanding how cells regulate their response to UV irradiation. UV irradiation triggers multiple cellular responses involving cell cycle arrest and repair of damage. We began by investigating how yeast cells regulate the enzymatic machinery that repairs UV damage via a process called nucleotide excision repair (NER). Like transcription, NER requires the coordinated activities of a large number of proteins, which assemble at particular chromatin sites. We have used a variety of molecular and genetic approaches, resulting in the discovery of a novel ubiquitin-mediated activity for a repair complex called NEF4. NEF4 possesses an ATPase in the Swi2/Snf2 ATPase family, but also functions as a ubiquitin ligase. This ubiquitin ligase activity is coordinated with other ubiquitin-mediated repair sub-pathways. We suggest that the dual functions for NEF4 in repair mediate turnover or remodeling of repair complex constituents at sites of damage in vivo. Subsequent analysis of NEF4 has led us to explore signal transduction pathways that detect and respond to UV damage at the transcriptional level.

Selected References

Dasgupta A, Darst RP, Martin KJ, Afshari CA, Auble DT. (2002) "Mot1 activates and represses transcription by direct, ATPase-dependent mechanisms." Proc Natl Acad Sci U S A. Mar 99:2666-71. [PubMed]

Ramsey KL, Smith JJ, Dasgupta A, Maqani N, Grant P, Auble DT. (2004) "The NEF4 complex regulates Rad4 levels and utilizes Snf2/Swi2-related ATPase activity for nucleotide excision repair." Mol Cell Biol. 24(14):6362-78. [PubMed]

Dasgupta A, Juedes SA, Sprouse RO, Auble DT. (2005) "Mot1-mediated control of transcription complex assembly and activity." EMBO J. May 24:1717-29. Epub 2005 Apr 7. [PubMed]

Sprouse RO, Brenowitz M, Auble DT. (2006) "Snf2/Swi2-related ATPase Mot1 drives displacement of TATA-binding protein by gripping DNA." EMBO J. Apr 25:1492-504. Epub 2006 Mar 16. [PubMed]

Dasgupta A, Sprouse RO, French S, Aprikian P, Hontz R, Juedes SA, Smith JS,Beyer AL, Auble DT. (2007) "Regulation of rRNA synthesis by TATA-binding protein-associated factor Mot1." Mol Cell Biol. 27:2886-96. Epub 2007 Feb 12. [PubMed]