SAGA is a transcriptional coactivator complex that is conserved across eukaryotes and performs multiple functions during transcriptional activation and elongation. One role is deubiquitination of histone H2B, and this activity resides in a distinct subcomplex called the deubiquitinating module (DUBm), which contains the ubiquitin-specific protease, Ubp8, bound to Sgf11, Sus1 and Sgf73. The deubiquitinating activity depends upon the presence of all four DUBm proteins. We report here the 1.90 Å resolution crystal structure of the DUB module bound to ubiquitin aldehyde, as well as the 2.45 Å resolution structure of the uncomplexed DUB module. The structure reveals an arrangement of protein domains that gives rise to a highly interconnected complex, which is stabilized by eight structural zinc atoms that are critical for enzymatic activity. The structure suggests a model for how interactions with the other DUBm proteins activate Ubp8, and allows us to speculate about how the DUB module binds to monoubiquitinated histone H2B in nucleosomes.

Catalysis by the RNase H-superfamily members is generally believed to require only two Mg2+ ions coordinated by active-site carboxylates. By examining the catalytic process of B. Halodurans RNase H1 in crystallo, however, we find that the two canonical Mg2+ ions and an additional K+ fail to align the nucleophilic water for RNA cleavage. Substrate alignment and product formation require a second K+ and a third Mg2+, which replaces the first K+ and departs immediately after cleavage. A third transient Mg2+ has also been observed for DNA synthesis, but there it coordinates the leaving group instead of the nucleophile as in this hydrolysis reaction. These transient cations have no contact with enzymes. Other DNA and RNA enzymes that catalyze consecutive cleavage and strand transfer reactions in a single active site may likewise require cation trafficking coordinated by substrate.

SUMMARY Transcription factor IIH (TFIIH) is essential for both transcription and nucleotide excision repair (NER). DNA lesions are initially detected by NER factors XPC and XPE or stalled RNA polymerases, but only bulky lesions are preferentially repaired by NER. To elucidate substrate specificity in NER, we have prepared homogeneous human ten-subunit TFIIH and its seven-subunit core (Core7) without the CAK module and show that bulky lesions in DNA inhibit the ATPase and helicase activities of both XPB and XPD in Core7 to promote NER, whereas non-genuine NER substrates have no such effect. Moreover, the NER factor XPA activates unwinding of normal DNA by Core7, but inhibits the Core7 helicase activity in the presence of bulky lesions. Finally, the CAK module inhibits DNA binding by TFIIH and thereby enhances XPC-dependent specific recruitment of TFIIH. Our results support a tripartite lesion verification mechanism involving XPC, TFIIH, and XPA for efficient NER.