The Saccharomyces cerevisiae RuvB-like Protein, Tih2p, Is Required for Cell Cycle Progression and RNA Polymerase II-directed Transcription

Abstract

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 ...

Control of transcription by Pontin and Reptin

“…Ino80 is recruited to at least two yeast promoters, and either activates or represses a large number of genes, presumably by mobilizing nucleosomes and altering the accessibility of the underlying DNA to the transcription machinery (19,20). As expected, Reptin is required for the correct expression of many of the same genes (20); other studies showed a significant overlap between the genes controlled by Pontin and those controlled by Reptin (18,(31)(32)(33). This demonstrates that Reptin and Pontin play a role in activation and repression and suggests that they function in the same complex (but see also the section on gene repression below).…”

Overexpression and role of the ATPase and putative DNA helicase RuvB-like 2 in human hepatocellular carcinoma

“…RUVBL2 was previously shown to be also required for the growth and survival of yeast. 7,31,32 Our data have shed some light on the mechanisms of apoptosis induced by RUVBL2 deficiency. We show that RUVBL2 down-regulation is associated with the increased conformational activation of the proapoptotic proteins Bak-1 and Bax.…”

“…Indeed, RUVBL2 has been found in supramolecular complexes that do not include ␤-catenin, 11 and ␤-catenin-independent functions of RUVBL2 have already been identified. 15 It is also noteworthy that RUVBL2 is required for the viability of yeast, 7,31,32 an organism devoid of ␤-catenin. In this context, 1 of the salient findings of this study was that in addition to its overexpression in HCC, RUVBL2 was also found in the cytoplasm of HCC cells.…”

TIP49, but not TRRAP, modulates c-Myc and E2F1 dependent apoptosis

“…Since neither Myc nor E2F exists in yeast, the nonviability of mammalian cells with a TRRAP knockout is unlikely to be due exclusively to defects in the Myc and E2F pathways. Temperature sensitive alleles of Rvb2(Tih2) causes a rapid G1 arrest and suppression of specific gene expression at the nonpermissive temperature, emphasizing the requirement for these ATPase/helicase proteins in transcription and cell cycle progression (Lim et al, 2000).…”