Regulation of transcription complex assembly and activity.

Mot1 is an essential yeast transcription factor that binds to the TATA-binding protein (TBP). TBP is essential for transcription in eukaryotes and the binding of TBP to promoters nucleates the assembly of the transcription complex. Below you can see a Jmol rendering of the TBP-DNA complex (TBP in green, DNA in blue). Mot1 uses ATP hydrolysis to remove TBP from DNA, and in so doing, exerts global effects on gene expression. We've learned quite a lot about how Mot1 recognizes TBP-DNA and what features of Mot1, TBP and DNA are required for this remarkable reaction to occur, but we don't understand yet how hydrolysis of ATP is used to drive the mechanical work of TBP-DNA disruption. This has been a terrific experimental system for providing general insight into how enzymes in this family work.

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The structure of the TBP-DNA complex guides our design and interpretation of biochemical studies. We use a variety of molecular, biochemical and biophysical approaches to analyze the Mot1-catalyzed TBP-DNA disruption reaction in vitro.  The complementary approaches available by studying this problem in living yeast cells allow us to determine if the in vitro observations are important in vivo, and likewise, the in vitro approaches allow us to determine mechanistic details that are not possible to visualize in vivo.

It is easy to imagine how Mot1-catalyzed removal of TBP from DNA leads to transcriptional repression, and our microarray analysis indicates that Mot1 functions in vivo to repress a number of genes. On the other hand, Mot1 also activates transcription of some genes, and our studies show that activation depends on the direct action of Mot1's ATPase at activated genes in vivo. Our results thus far suggest that one mechanism of Mot1-mediated activation involves Mot1's ability to dismantle transcriptionally incompetent complexes that form on certain promoters. Our hypothesis is that when Mot1 disassembles such complexes, the promoter has another opportunity for a functional transcription complex to be assembled there. We're following up these observations by determining what the nature of the inactive transcription complexes is and why they form on certain promoters. Mot1 may also have a novel activity at activated promoters that we don't understand yet. We use a variety of genetic and molecular approaches to gain insight into transcription complex assembly and dynamics in vivo.

Mechanism and Regulation of Nucleotide excision repair (NER)

NER is a complicated repair pathway in eukaryotes comprised of machinery that recognizes and removes thymine dimers (induced by UV irradiation) and a wide variety of bulky DNA adducts (induced by reactive chemicals in the environment).  One rather mysterious factor required for NER is called NEF4 (nucleotide excision repair factor 4).  NEF4 is required for repair of nontranscribed DNA, including the nontranscribed strand of transcribed genes.  One subunit of NEF4 is called Rad16, which is a member of the Snf2/Swi2 ATPase family.   NEF4 displays ATP-dependent binding to damaged DNA, and one model is that ATP hydrolysis by NEF4 allows the complex to translocate along DNA to scan for DNA damage.  Rad16 can be functionally grouped with a subset of Snf2/Swi2-related ATPases because it also has a RING domain located between blocks of conserved sequences in the ATPase.

Rad16 is physically associated with a leucine-rich repeat (LRR) protein called Rad7.  Our analysis of Rad7 led to the surprising observation that Rad7 also has an F-box motif.  The yeast homolog of human Elongin C, Elc1, is also a component of NEF4.  Complexes with RING domain subunits, F-box subunits, and/or Elongin C function as ubiquitin ligases (E3s), adaptors that determine which substrates become covalently modified with ubiquitin (or ubiquitin-related proteins).  Ubiquitylation of proteins can function in signaling or target proteins for proteolysis by the proteasome.  Based on the presence of these motifs in subunits of NEF4, we proposed that NEF4 functions as both an ATP-driven DNA translocating enzyme and as an E3.  Extensive mutational analysis provides strong support for the importance of Elc1, RING domain, and F-box in NEF4 function.  Genetic analysis has also identified other components of the NEF4 repair pathway.  The levels of the DNA damage recognition factor Rad4 are controlled by NEF4, suggesting that the NEF4 E3 activity is involved in regulating NER capacity in vivo by regulating the levels of Rad4.  Interestingly, deregulation of Elongin C has been associated with certain cancers, and it is possible that alteration of a human NEF4 activity causes genomic instability in human cells.  Using a variety of approaches we are dissecting this novel repair pathway in vivo and we are employing similar strategies to those we have used with Mot1 to better define the function of NEF4 in vivo and in vitro.