We previously reported that TIP49a is a novel mammalian DNA helicase showing structural similarity with the bacterial recombination factor RuvB. In this study, we isolated a new TIP49a-related gene, termed TIP49b, from human and yeast cells. TIP49b also resembled RuvB, thus suggesting that TIP49a and TIP49b are included in a gene family. Like TIP49a, TIP49b was abundantly expressed in the testis and thymus. Enzyme assays revealed that TIP49b was an single-stranded DNAstimulated ATPase and ATP-dependent DNA helicase. Most of the enzymatic properties of TIP49b were the same as those of TIP49a, whereas the polarity of TIP49b DNA helicase activity (5 to 3) was the opposite to that of TIP49a. TIP49b and TIP49a bound to each other and were included in the same complex of ϳ700 kDa in a cell. We found that TIP49b was an essential gene for the growth of Saccharomyces cerevisiae, as is the TIP49a gene, suggesting that TIP49b does not complement the TIP49a function and vice versa. From these observations, we suggest that TIP49b plays an essential role in the cellular processes involved in DNA metabolism.

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We have isolated a novel nuclear protein with a molecular mass of 49 kDa (TIP49a) from rat liver. The rat TIP49a showed structural resemblance to several bacterial RuvBs and also displayed Walker A and B motifs. We overproduced the recombinant TIP49a in Escherichia coli and purified it to near homogeneity. Biochemical investigations demonstrated that TIP49a possessed ATPase activity that was stimulated by single-stranded DNA but neither by double-stranded DNA nor by any forms of RNA polymers tested. Moreover, a UV crosslinking assay indicated TIP49a specifically interacted with ATP. Interestingly, we found that DNA duplex was unwound by the recombinant TIP49a in the presence of ATP or dATP. Optimal concentrations of ATP and Mg 2؉for the helicase activity were 1-2 mM and 0.25-1 mM, respectively. Displacement of the DNA strand occurred in the 3 to 5 direction with respect to the singlestranded DNA flanking the duplex. Western blot analysis revealed that TIP49a was abundantly expressed in testes and moderately in spleen, thymus, and lung. In mouse seminiferous tubules, the protein was restrictively observed in germ lineages from late pachytene spermatocytes to round spermatids. From these observations, we propose that TIP49a is a novel DNA helicase and may play a role in nuclear processes such as recombination and transcription.The unwinding of parent DNA strands is a prerequisite to basic genetic processes including DNA replication, DNA repair, recombination, and transcription (1, 2). In each of these processes, unwinding of duplex DNA is catalyzed by a DNA helicase, which functions to destabilize hydrogen bonds between complementary base pairs in duplex DNA. The energy necessary for this reaction is provided by hydrolysis of nucleosides and deoxynucleoside 5Ј-triphosphates, meaning that DNA helicase generally possesses an intrinsic ATPase activity (2).DNA helicases are found ubiquitously from prokaryotes to eukaryotes. Biochemical studies and computer analysis have revealed that helicases have several characteristic motifs and that some of them are functionally important. Two sequence signatures, so-called Walker A and B motifs, have been identified in all helicases examined and in a wide variety of other NTP-utilizing enzymes (3, 4). On the basis of the primary sequence analysis of the Saccharomyces cerevisiae genome, there are at least 41 helicase genes in the genome (5). However, most of our biochemical knowledge about DNA helicases comes from analyses of bacterial and phage enzymes (1, 2), because little is known about the biochemical properties of most of the eukaryotic DNA helicases. However, recent studies demonstrated that six established or putative helicases are mutated in the human diseases such as xeroderma pigmentosum, Bloom's syndrome, Cockayne's syndrome, trichothiodystrophy, Werner's syndrome, and ␣-thalassemia (5). Thus, identification of a novel helicase is medically important.We originally identified a 49-kDa TATA-binding protein (TBP) 1 -interacting protein (TIP49a; termed TIP49 in previous re...

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RNA, but not by poly(A)؊ RNA. RNA transcribed in vitro from cDNA encoding a b-Zip protein could stimulate the ATPase activity. This is the first report to demonstrate a specific RNA requirement for ATPase with respect to the proteasomal ATPases. Our present work suggests that SUG1 can specifically interact with protein-coding RNA (mRNA) and play some roles in mRNA metabolism.The 26 S proteasome is a huge protease complex that degrades short-lived proteins related to metabolic regulation and cell cycle progression (1, 2). It is composed of the 20 S catalytic core and an ATPase-containing 22 S regulatory complex (3, 4). The human 22 S complex contains at least five highly related putative ATPases, i.e. TBP1, TBP7, S4, MSS1, and p45 (a homolog of yeast SUG1), which are members of a novel ATPase family named the AAA family (ATPases associated with a variety of cellular activities) (5). The family members have a highly conserved ATPase module with 200 amino acids and fulfill a large diversity of functions. It was demonstrated that the five ATPases contain four conserved motifs characteristic of putative ATP-dependent RNA/DNA helicases (6 -8). We previously reported constant distances between each motif among proteasomal ATPases, suggesting the functional importance of these motifs (6).Of the above ATPases, SUG1 (11) has been shown by various studies to be involved in transcriptional regulation in addition to proteolytic function. The yeast SUG1 was reported to be a component of the RNA polymerase II (pol II) holoenzyme (12, 13), and Trip1, a human homolog of the SUG1, interacts with the thyroid hormone receptor (14). Baur et al. (15) also reported that mouse SUG1 interacts with various nuclear receptors. However, it is still controversial as to whether or not SUG1 acts as an intrinsic transcription factor because one cannot eliminate the possibility that those ATPases are involved in degradation of transcription factors mediated by the 26 S proteasome. Actually, some transcription factors are regulated by proteasome-dependent proteolysis (1). Biochemical study is required to resolve issues such as how the ATPases are involved in RNA metabolism such as transcriptional regulation. In this study, we report the purification and characterization of rat SUG1. We found that rSUG1 1 exhibited ATPase activity that was specifically stimulated by particular RNA molecules. EXPERIMENTAL PROCEDURESExpression and Purification of Rat SUG1-SUG1 cDNA was cloned from a rat liver cDNA library as described previously (6). Histidinetagged rSUG1 was overexpressed in Escherichia coli by use of the pET vector system (16). Insoluble recombinant rSUG1 was purified by Ni 2ϩ -agarose under denaturing conditions according to the instructions supplied by Qiagen. The resulting proteins were further subjected to a preparative SDS-PAGE and recovered from the excised gels. The protein was redissolved in a urea-containing buffer (25 mM Tris-HCl (pH 7.5), 0.3 M NaCl, 1 mM 2-mercaptoethanol, 0.1% Nonidet P-40, 10% glycerol, and 8 M urea), and the urea w...

TIP49a (just called as simply TIP49 in previous reports [Kanemaki et al., 1997; Makino et al., 1998]) was found in a rat nuclear protein complex that included the TATA-binding protein. TIP49a possesses multiple sequence motifs for ATPase and DNA helicase. Since TIP49a structurally resembles prokaryotic DNA helicase RuvB, TIP49a is resumed to be a putative DNA helicase. We demonstrated TIP49a-related gene(s) in variety organisms from human to archaea. Amino acid identities expressed as aligned scores of human, yeast, and A. fulgidus TIP49a gene counterparts to the rat sequence were 99, 67, and 46, respectively. Strikingly, two homologous regions of mammalian TIP49a and bacterial RuvB exhibited an aligned score of 17-38. We demonstrated that the eukaryotic TIP49a counterparts were immunologically conserved. These lines of evidence show that the TIP49a gene is a notable example of a highly conserved gene among organisms. An extensive homology search revealed another class of TIP49-related gene in the eukaryotes, designated as TIP49b. Moreover, a phylogenetical study suggested that archaeal TIP49 genes belong to the TIP49b ancestor but not to the TIP49a one and that TIP49a evolved from TIP49b in accordance with divergence of archaea and eukarya. The TIP49 gene family is thought to play a fundamental role in a biological activity.

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TBP-interacting protein 49 (TIP49) was originally identified as a TBP-binding protein, and two related proteins are encoded by individual genes, tip49a and b. Although the function of this gene family has not been elucidated, they are supposed to play a critical role in nuclear events because they interact with various kinds of nuclear factors and have DNA helicase activities. At least, TIP49a has been suggested to act as an autoantigen in some patients with autoimmune diseases. In this study, we investigated the chromosome positions of this family of genes. Human tip49a and tip49b genes were mapped on 3q21 and 19q13.2, respectively. Consistent with the notion that tip49 family genes are essential for cell growth, Northern blot analysis demonstrated that both genes are expressed ubiquitously in human tissues. It is worthy of notice that the testes contained large amounts of the both transcripts. These results are consistent with our previous results from tissue distribution analysis for of TIP49 proteins.