Abstract
The Ssl1/p44 subunit is a core component of the yeast/mammalian general transcription factor TFIIH, which is involved in transcription and DNA repair. Ssl1/p44 binds to and stimulates the Rad3/XPD helicase activity of TFIIH. To understand the helicase stimulatory mechanism of Ssl1/p44, we determined the crystal structure of the N-terminal regulatory domain of Ssl1 from Saccharomyces cerevisiae. Ssl1 forms a von Willebrand factor A fold in which a central six-stranded β-sheet is sandwiched between three α helices on both sides. Structural and biochemical analyses of Ssl1/p44 revealed that the β4-α5 loop, which is frequently found at the interface between von Willebrand factor A family proteins and cellular counterparts, is critical for the stimulation of Rad3/XPD. Yeast genetics analyses showed that double mutation of Leu-239 and Ser-240 in the β4-α5 loop of Ssl1 leads to lethality of a yeast strain, demonstrating the importance of the Rad3-Ssl1 interactions to cell viability. Here, we provide a structural model for the Rad3/XPD-Ssl1/p44 complex and insights into how the binding of Ssl1/p44 contributes to the helicase activity of Rad3/XPD and cell viability.
Highlights
Ssl1/p44 is a critical helicase stimulating factor for Rad3/XPD
Structural and biochemical analyses of Ssl1/p44 revealed that the 4-␣5 loop, which is frequently found at the interface between von Willebrand factor A family proteins and cellular counterparts, is critical for the stimulation of Rad3/XPD
We show that the 4-␣5 loop of Ssl1/p44 is important for the binding and stimulation of Rad3/XPD, and the interaction between Rad3/XPD and Ssl1/p44 is critical for cell viability
Summary
Results: Structure-based mutation analysis of the 4-␣5 loop of Ssl1/p44 resulted in defects of Rad3/XPD stimulation and yeast cell lethality. We provide a structural model for the Rad3/XPD-Ssl1/p44 complex and insights into how the binding of Ssl1/p44 contributes to the helicase activity of Rad3/XPD and cell viability. The structures of only a limited number of individual components are available, which hampers the modeling of complete TFIIH It is unclear how Ssl1/p44 interacts with and stimulates Rad3/XPD helicase activity and how the disease-causing C-terminal mutations of Rad3/XPD are correlated with its interaction with Ssl1/p44. To address these questions, we have determined the crystal structure of the N-terminal regulatory domain of Saccharomyces cerevisiae Ssl. Based on the derived structure, mutation analysis, and genetics studies, we provide a model for the Rad3/XPDSsl1/p44 subcomplex
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