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

Wollmann P, Cui S, Viswanathan R, Berninghausen O, Wells MN, Moldt M, Witte G,Butryn A, Wendler P, Beckmann R, Auble DT, Hopfner KP. (2011) "Structure and mechanism of the Swi2/Snf2 remodeller Mot1 in complex with its substrate TBP." Nature. Jul 475(7356):403-7. [PubMed]

Poorey K, Sprouse RO, Wells MN, Viswanathan R, Bekiranov S, Auble DT. (2010) "RNA synthesis precision is regulated by preinitiation complex turnover." Genome Res. 20(12):1679-88. Epub 2010 Sep 20. [PubMed]

Wade SL, Poorey K, Bekiranov S, Auble DT. (2009) "The Snf1 kinase and proteasome-associated Rad23 regulate UV-responsive gene expression." EMBO J. Oct 28(19):2919-31. Epub 2009 Aug 13. [PubMed]

Sprouse RO, Wells MN, Auble DT. (2009) "TATA-binding protein variants that bypass the requirement for Mot1 in vivo." J Biol Chem. Feb 284:4525-35. Epub 2008 Dec 21. [PubMed]

Auble DT. (2009) "The dynamic personality of TATA-binding protein." Trends Biochem Sci. 34:49-52. Epub 2008 Nov 27. [PubMed]

Sprouse RO, Karpova TS, Mueller F, Dasgupta A, McNally JG, Auble DT. (2008) "Regulation of TATA-binding protein dynamics in living yeast cells." Proc Natl Acad Sci U S A. Sep 105(36):13304-8. Epub 2008 Sep 2. [PubMed]

Sprouse RO, Shcherbakova I, Cheng H, Jamison E, Brenowitz M, Auble DT. (2008) "Function and structural organization of Mot1 bound to a natural target promoter." J Biol Chem. Sep 283(36):24935-48. Epub 2008 Jul 7. [PubMed]