The TATA binding protein (TBP) is a central component of the eukaryotic transcription machinery and is subjected to both positive and negative regulation. As is evident from structural and functional studies, TBP's concave DNA binding surface is inhibited by a number of potential mechanisms, including homodimerization and binding to the TAND domain of the TFIID subunit TAF1 (yTAF II 145/130). Here we further characterized these interactions by creating mutations at 24 amino acids within the Saccharomyces cerevisiae TBP crystallographic dimer interface. These mutants are impaired for dimerization, TAF1 TAND binding, and TATA binding to an extent that is consistent with the crystal or nuclear magnetic resonance structure of these or related interactions. In vivo, these mutants displayed a variety of phenotypes, the severity of which correlated with relative dimer instability in vitro. The phenotypes included a low steady-state level of the mutant TBP, transcriptional derepression, dominant slow growth (partial toxicity), and synthetic toxicity in combination with a deletion of the TAF1 TAND domain. These phenotypes cannot be accounted for by defective interactions with other known TBP inhibitors and likely reflect defects in TBP dimerization.Activation of eukaryotic genes is a multistep process, involving the coalescence of promoter-specific activators, chromatinremodeling complexes, and components of the general transcription machinery at promoters. An important part of the activation process is the removal of inhibitors associated with latent activators, promoters, and the general transcription machinery. One component of the general transcription machinery that is subjected to substantial inhibition is the TATA binding protein (TBP) (reviewed in reference 75). Virtually all genes require TBP for function, and its association with promoters is generally linked to transcriptional activity (57, 63). Preventing unregulated promoter binding by TBP may be critical for preventing unregulated gene expression. TBP access might be prevented in part by nucleosome formation over the TATA box (43, 80). However, many quiescent genes are not derepressed upon histone depletion (83), indicating that other inhibitory mechanisms might prevent TBP from binding to promoters.A number of proteins inhibit TBP function. These include the TAF1 (yTAF II 145/130) subunit of TFIID, NC2, Mot1, the Spt3/Spt8 subunits of SAGA, the Ccr4-Not complex, and a second molecule of TBP in the form of homodimers. Here we focus on two inhibitory interactions which are directed at TBP's concave surface: TBP dimerization and the TAF1 TAND domain.TFIID is a multisubunit complex consisting of TBP and TAFs (19,77,78). TFIID is required for activated transcription but is intrinsically inhibitory toward TBP-TATA interactions (78). At least part of this inhibitory activity might reside within the amino-terminal TAND domain of the TFIID subunit, TAF1 (11,52,70). Mutagenesis studies have delineated Drosophila and yeast TANDs as two subdomains, I and II (52, 54). ...

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TFIIH is indispensable for nucleotide excision repair (NER) and RNA polymerase II transcription. Its tenth subunit was recently discovered in yeast as Tfb5. Unlike other TFIIH subunits, Tfb5 is not essential for cell survival. We have analyzed the role of Tfb5 in NER. NER was deficient in the tfb5 deletion mutant cell extracts, and was specifically complemented by purified Tfb5 protein. In contrast to the extreme ultraviolet (UV) sensitivity of rad14 mutant cells that lack any NER activity, tfb5 deletion mutant cells were moderately sensitive to UV radiation, resembling that of the tfb1-101 mutant cells in which TFIIH activity is compromised but not eliminated. Thus, Tfb5 protein directly participates in NER and is an accessory NER protein that stimulates the repair to the proficient level. Lacking a DNA binding activity, Tfb5 was found to interact with the core TFIIH subunit Tfb2, but not with other NER proteins. The Tfb5–Tfb2 interaction was correlated with the cellular NER function of Tfb5, supporting the functional importance of this interaction. Our results led to a model in which Tfb5 acts as an architectural stabilizer conferring structural rigidity to the core TFIIH such that the complex is maintained in its functional architecture.

Nucleotide excision repair (NER) is a major cellular defense mechanism against DNA damage. We have investigated the role of Mms19 in NER in the yeast Saccharomyces cerevisiae. NER was deficient in the mms19 deletion mutant cell extracts, which was complemented by the NER/transcription factor TFIIH, but not by purified Mms19 protein. In mms19 mutant cells, protein levels of the core TFIIH component Rad3 (XPD homologue) and Ssl2 (XPB homologue) were significantly reduced by up to 3.5-and 2.2-fold, respectively. The other four essential subunits of the core TFIIH, Tfb1, Tfb2, Ssl1, and Tfb4, and the TFIIK subunits Tfb3, Kin28, and Ccl1 of the holo TFIIH were not much affected by Mms19. Elevating Rad3 protein concentration by overexpressing the protein from a plasmid under the GAL1 promoter control restored proficient NER in mms19 mutant cells, as indicated by complementation for UV sensitivity. Overexpression of Ssl2 had no effect on repair. Overexpression of Rad3, Ssl2, or both proteins, however, could not correct the temperature-sensitive growth defect of mms19 mutant cells. These results show that Mms19 functions in NER by sustaining an adequate cellular concentration of the TFIIH component Rad3 and suggest that Mms19 has distinct and separable functions in NER and cell growth, thus implicating Mms19 protein as a novel multifunctional regulator in cells.DNA damage ͉ DNA repair ͉ repair regulation ͉ transcription factor

The TATA-binding protein (TBP) plays a central role in assembling eukaryotic transcription complexes and is subjected to extensive regulation including auto-inhibition of its DNA binding activity through dimerization. Previously, we have shown that mutations that disrupt TBP dimers in vitro have three detectable phenotypes in vivo, including decreased steady-state levels of the mutants, transcriptional derepression, and toxicity toward cell growth. In an effort to more precisely define the multimeric structure of TBP in vivo, the crystallographic dimer structure was used to design mutations that might enhance dimer stability. These mutations were found to enhance dimer stability in vitro and significantly suppress in vivo phenotypes arising from a dimer-destabilizing mutation. Although it is conceivable that phenotypes associated with dimer-destabilizing mutants could arise through defective interactions with other cellular factors, intragenic suppression of these phenotypes by mutations designed to stabilize dimers provides compelling evidence for a crystallographic dimer configuration in vivo.Gene expression levels are derived from a net output of a dynamic interplay of positive and negative regulatory factors. The main steps leading toward gene activation include chromatin modification and remodeling, TBP 1 delivery, and assembly of the RNA polymerase II holoenzyme (1-3). Each of these steps is the target of multiple regulatory factors. TBP delivery to promoters is directed by the positive action of transcriptional activators, acetylated histone tails, SAGA and TFIID delivery complexes, basal factors such as TFIIA and TFIIB, and the TATA box. Counteracting this delivery are Mot1, NC2, a portion of TAF1 (termed TAND), the amino-terminal domain of TBP, and TBP auto-inhibition, which occurs through dimerization and occlusion of its DNA binding surface.TBP auto-inhibition through dimerization is an evolutionary conserved process, occurring from yeast to mammals (4 -15). The structure of TBP dimers has been defined crystallographically and through biochemical analysis. Dimer instability caused by mutations along the crystallographic dimer interface correlate with transcriptional derepression in yeast cells (8,15), and this derepression occurs genome-wide at about 7% of all genes (16). Together, these findings indicated that TBP dimerization represents a physiologically important mechanism for auto-inhibiting its DNA binding activity. Nonetheless, the notion of an autoinhibited TBP dimer has been sufficiently controversial (17) that we pursued the possibility of using the x-ray crystal structure of TBP dimers to design stabilizing mutations that might intragenically suppress a dimerization mutant.We focused on two residues, Arg-98 and Arg-171, which lie on opposite sides of a TBP monomer. In the dimer configuration, Arg-98 of one monomer lies immediately across Arg-171 of the opposing monomer (11). We reasoned that changing one or the other to an acidic residue might generate a positive electrostatic interaction wh...

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DNA damage tolerance is a major cellular mechanism in response to unrepaired DNA lesions during replication. It allows replication to completion in the presence of DNA lesions that block replicative polymerases. Damage tolerance consists of two major mechanisms: template switching and translesion DNA synthesis. Translesion synthesis directly copies damaged sites of the DNA template during replication. Consequently, error-prone translesion synthesis constitutes the major mechanism of base damage-induced mutagenesis. In eukaryotes, translesion synthesis is carried out by the Rev1-Polα pathway. Rev1 possesses a dCMP transferase activity, and is a member of the Y family of DNA polymerases. Additionally, it is widely believed that Rev1 plays non-catalytic functions in translesion synthesis. However, the non-catalytic function of Rev1 is not clearly defined. Using the yeast model system, we have investigated non-catalytic functions of Rev1 in translesion synthesis and base damage-induced mutagenesis. Specifically, we examined the role of Rev1-Rad5 interaction in translesion synthesis of UV damage and a site-specific 1, N6-ethenoadenine (a DNA lesion of lipid peroxidation products). Although Rad5 is better known for its role in template switching, it stably interacts with Rev1. Interaction domains in the Rev1 and Rad5 proteins were defined. Translesion synthesis and mutagenesis required Rad5 in cells. Disrupting the Rev1-Rad5 interaction by mutating Rev1 did not affect the dCMP transferase of Rev1, but led to inactivation of the translesion synthesis pathway. These results show that Rad5 is an indispensible component of the Rev1-Pol ≤ pathway for translesion synthesis and base damage-induced mutagenesis, and a non-catalytic function of Rev1 in translesion DNA synthesis is mediated by its stable interaction with Rad5. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2553. doi:1538-7445.AM2012-2553