Two highly conserved RuvB-like putative DNA helicases, p47/TIP49b and p50/TIP49a, have been identified in the eukaryotes. Here, we study the function of Saccharomyces cerevisiae TIH2, which corresponds to mammalian p47/TIP49b. Tih2p is required for vegetative cell growth and localizes in the nucleus. Immunoprecipitation analysis revealed that Tih2p tightly interacts with Tih1p, the counterpart of mammalian p50/TIP49a, which has been shown to interact with the TATA-binding protein and the RNA polymerase II holoenzyme complex. Furthermore, the mutational study of the Walker A motif, which is required for nucleotide binding and hydrolysis, showed that this motif plays indispensable roles in the function of Tih2p. When a temperature-sensitive tih2 mutant, tih2-160, was incubated at the nonpermissive temperature, cells were rapidly arrested in the G 1 phase. Northern blot analysis revealed that Tih2p is required for transcription of G 1 cyclin and of several ribosomal protein genes. The similarities between the mutant phenotypes of tih2-160 and those of taf145 mutants suggest a role for TIH2 in the regulation of RNA polymerase II-directed transcription.One of the common post-translational modifications of nuclear and cytosolic proteins in eukaryotes is the addition of N-acetylglucosamine residues O-linked (O-GlcNAc) 1 to serine and threonine residues. A number of physiologically or structurally important proteins have thus far been shown to contain O-GlcNAc, including the largest subunit of RNA polymerase II (RNAP II) (1), transcription factors such as Sp1 and hepatocyte nuclear factor 1 (2-4), nuclear pore proteins (5, 6) and chromatin-associated proteins (7). To study nuclear factors involved in nuclear transport and other nuclear events, we previously performed an in vitro binding assay of a rat liver nuclear matrix fraction using a wheat germ agglutinin affinity column (8).Several O-GlcNAc-containing proteins such as nucleoporins as well as nonglycosylated proteins like importin ␤ were isolated (9). Two RuvB-like proteins, p50 (9) and p47 (10), were also isolated by this method probably because of their interaction with O-GlcNAc-bearing proteins. RuvB is a prokaryotic DNA helicase, and its helicase activity and DNA binding affinity are enhanced by interaction with RuvA (11,12). These two factors form a large motor protein complex to promote branch migration at Holliday junctions at the late stages of homologous recombination. The p50/p47 and RuvB proteins share highly conserved Walker domains (A and B), indicative of proteins that bind nucleotide triphosphates (10, 13). Based on this, p50 and p47 were proposed to be the eukaryotic homologues of the RuvB DNA helicase. Indeed, p50 and p47 are present in a wide range of eukaryotes ranging from yeast to mammals, suggesting that this basic helicase activity may be conserved among the eukaryotes.In addition to recombination, transient unwinding of the DNA duplex is required in many cellular processes such as replication, transcription, and DNA repair. To accommodate ...

In Saccharomyces cerevisiae, two highly conserved proteins, Rvb1p/Tih1p and Rvb2p/Tih2p, have been demonstrated to be major components of the chromatinremodeling INO80 complex. The mammalian orthologues of these two proteins have been shown to physically associate with the TATA-binding protein (TBP) in vitro but not clearly in vivo. Here we show that yeast proteins interact with TBP under both conditions. To assess the functional importance of these interactions, we examined the effect of mutating both TIH2/ RVB2 and SPT15, which encodes TBP, on yeast cell growth. Intriguingly, only those spt15 mutations that affected the ability of TBP to bind to the TATA box caused synthetic growth defects in a tih2-ts160 background. This suggests that Tih2p might be important in recruiting TBP to the promoter. A DNA microarray technique was used to identify genes differentially expressed in the tih2-ts160 strain grown at the restrictive temperature. Only 34 genes were significantly and reproducibly affected; some up-regulated and others down-regulated. We compared the transcription of several of these Tih2p target genes in both wild type and various mutant backgrounds. We found that the transcription of some genes depends on functions possessed by both Tih2p and TBP and that these functions are substantially impaired in the spt15/tih2-ts160 double mutants that confer synthetic growth defects.