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• W2023489445 abstract "A human protein (RUVBL1), consisting of 456 amino acids (50 kDa) and highly homologous to RuvB, was identified by using the 14-kDa subunit of replication protein A (hsRPA3) as bait in a yeast two-hybrid system. RuvB is a bacterial protein involved in genetic recombination that bears structural similarity to subunits of the RF-C clamp loader family of proteins. Fluorescence in situhybridization analysis demonstrated that the RUVBL1 gene is located at 3q21, a region with frequent rearrangements in different types of leukemia and solid tumors. RUVBL1 co-immunoprecipitated with at least three other unidentified cellular proteins and was detected in the RNA polymerase II holoenzyme complex purified over multiple chromatographic steps. In addition, two yeast homologs, scRUVBL1 and scRUVBL2 with 70 and 42% identity to RUVBL1, respectively, were revealed by screening the complete Saccharomyces cerevisiae genome sequence. Yeast with a null mutation in scRUVBL1 was nonviable. Thus RUVBL1 is an eukaryotic member of the RuvB/clamp loader family of structurally related proteins from bacteria and eukaryotes that is essential for viability of yeast. A human protein (RUVBL1), consisting of 456 amino acids (50 kDa) and highly homologous to RuvB, was identified by using the 14-kDa subunit of replication protein A (hsRPA3) as bait in a yeast two-hybrid system. RuvB is a bacterial protein involved in genetic recombination that bears structural similarity to subunits of the RF-C clamp loader family of proteins. Fluorescence in situhybridization analysis demonstrated that the RUVBL1 gene is located at 3q21, a region with frequent rearrangements in different types of leukemia and solid tumors. RUVBL1 co-immunoprecipitated with at least three other unidentified cellular proteins and was detected in the RNA polymerase II holoenzyme complex purified over multiple chromatographic steps. In addition, two yeast homologs, scRUVBL1 and scRUVBL2 with 70 and 42% identity to RUVBL1, respectively, were revealed by screening the complete Saccharomyces cerevisiae genome sequence. Yeast with a null mutation in scRUVBL1 was nonviable. Thus RUVBL1 is an eukaryotic member of the RuvB/clamp loader family of structurally related proteins from bacteria and eukaryotes that is essential for viability of yeast. replication protein A Homo sorpiens RNA polymerase II kilobase pair(s) dithiothreitol phenylmethylsulfonyl fluoride polyacrylamide gel electrophoresis glyceraldehyde-3-phosphate dehydrogenase glutathioneS-transferase CREB-binding protein base pair(s). Genetic recombination plays a critical role in maintaining gene diversification through chromosomal rearrangement and also genome stability through the repair of DNA damage. The activities of many proteins are required for recombination. In bacteria, for instance, RecA protein with the assistance of single-stranded DNA-binding protein promotes strand exchange with a homologous duplex and creates a four-strand intermediate or Holliday junction. The latter is then translocated by RuvA and RuvB proteins through branch migration and resolved by RuvC protein to yield recombinant DNA products (1Eggleston A.K. Mitchell A.H. West S.C. Cell. 1997; 89: 607-617Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). RuvB protein is a DNA-dependent ATPase and helicase that forms hexameric rings and has a low intrinsic affinity for DNA. RuvA is a structure-specific DNA-binding protein that has a high affinity for Holliday junctions and interacts with RuvB to form specific complexes with Holliday junctions. The presence of RuvA facilitates RuvB-mediated ATP hydrolysis and branch migration (2Tsaneva I.R. Muller N. West S.C. Cell. 1992; 69: 1171-1180Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 3Muller B. Tsaneva I.R. West S.C. J. Biol. Chem. 1993; 268: 17179-17184Abstract Full Text PDF PubMed Google Scholar). Recombination activity has also been identified in eukaryotes and may be related to cell cycle progression. The Holliday intermediates in yeast accumulate to the highest level and become detectable during S phase (4Zou H. Rothstein R. Cell. 1997; 90: 87-96Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). A yeast homolog of bacterial RecA, Rad51, is essential for spore formation during meiosis (5Shinohara A. Ogawa H. Ogawa T. Cell. 1992; 69: 457-470Abstract Full Text PDF PubMed Scopus (1052) Google Scholar). Rad51 mRNA is significantly increased during meiosis and is also regulated during the mitotic cell cycle, with the highest levels found at the G1/S boundary (6Flygare J. Benson F. Hellgren D. Biochim. Biophys. Acta. 1996; 1312: 231-236Crossref PubMed Scopus (68) Google Scholar, 7Yamamoto A. Taki T. Yagi H. Habu T. Yoshida K. Yoshimura Y. Yamamoto K. Matsushiro A. Nishimune Y. Morita T. Mol. Gen. Genet. 1996; 251: 1-12Crossref PubMed Google Scholar). Homologs of bacterial RecA are also found in other eukaryotes, including Xenopus laevis, Lilium longiflorum, Neurospora crassa, Arabidopsis thaliana, mouse, chicken, and man (8Gupta R.C. Bazenore L.R. Bolub E.I. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 463-468Crossref PubMed Scopus (239) Google Scholar), suggesting that the machinery involved in recombination is highly conserved among all organisms from bacteria to man. Consistently, single-stranded DNA-binding protein is also functionally conserved through evolution. Human single-stranded DNA-binding protein, also known as human replication protein A (hsRPA),1 is a heterotrimer of 70, 32, and 14 kDa subunits. In both man and yeast, RPA serves as an important accessory factor in pairing and strand exchange carried out by Rad51 (9Sung P. Robberson D.L. Cell. 1995; 82: 453-461Abstract Full Text PDF PubMed Scopus (425) Google Scholar, 10Baumann P. West S.C. EMBO J. 1997; 16: 5198-5206Crossref PubMed Scopus (119) Google Scholar). Recent evidence suggests that recombination proteins may be physically associated with proteins involved in transcription. Tumor suppressor p53, a transcriptional activator for many important genes, has been demonstrated to interact with hsRPA (11Dutta A. Ruppert J.M. Aster J.C. Winchester E. Nature. 1993; 365: 79-82Crossref PubMed Scopus (331) Google Scholar) and with hRad51 to inhibit the activities of hRad51 in recombination (12Sturzbecher H.W. Donzelmann B. Henning W. Knippschild U. Buchhop S. EMBO J. 1996; 15: 1992-2002Crossref PubMed Scopus (332) Google Scholar). Tumor suppressor BRCA1 co-localizes at nuclear foci with hRad51 during S phase and co-immunoprecipitates with the same (13Scully R. Chen J. Plug A. Xiao Y. Weaver D. Feunteun J. Ashley T. Livingston D.M. Cell. 1997; 88: 265-275Abstract Full Text Full Text PDF PubMed Scopus (1328) Google Scholar). However, BRCA1 is also a component of the RNA polymerase II holoenzyme (14Scully R. Anderson S.F. Chao D.M. Wei W. Ye L. Young R.A. Livingston D.M. Parvin J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5605-5610Crossref PubMed Scopus (425) Google Scholar) and has been implicated in transcriptional activation (15Samasundaram K. Zhang H. Zeng Y.X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (472) Google Scholar). Thus proteins like Rad51 and RPA, likely involved in recombination, physically interact with proteins involved in transcription. In this paper, we report the identification of a human protein (RUVBL1) related in sequence to bacterial RuvB by using the 14-kDa subunit of human RPA (hsRPA3) as bait in a yeast two-hybrid system. The RUVBL1 gene is mapped to 3q21, a region with frequent rearrangements in different types of leukemia and solid tumors (16Kashuba V.I. Gizatullin R.Z. Protopopov A.I. Allikmets R. Korolev S. Li J. Boldog F. Tory K. Zabarovska V. Marcsek Z. Sumegi J. Klein G. Zabarovsky E.R. Kisselev L. FEBS Lett. 1997; 419: 181-185Crossref PubMed Scopus (37) Google Scholar, 17Rynditch A. Pekarsky Y. Schnittger S. Gardiner K. Gene (Amst.). 1997; 193: 49-57Crossref PubMed Scopus (31) Google Scholar). About 30% of the total cellular RUVBL1 co-purifies with the RNA polymerase II holoenzyme over multiple chromatographic steps. In addition, two yeast homologs scRUVBL1 (GenBankTM accession number S52968) and scRUVBL2 (GenBankTM accession number S61029) with 70 and 42% identity to RUVBL1, respectively, are revealed by screening the complete Saccharomyces cerevisiae genome sequence. Knockout of scRUVBL1 demonstrates that scRUVBL1 is essential for growth. Thus, RUVBL1 is an essential protein (in yeast) and is partly present in the RNA polymerase II holoenzyme complex. pAS-hsRPA3 was constructed by transferring the EcoRI-XhoI fragment of pEGRPA3 (18Lin Y.-L. Chen C. Keshav K.F. Winchester E. Dutta A. J. Biol. Chem. 1996; 271: 17190-17198Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) to pAS2 (CLONTECH). hsRPA3 was expressed as a fusion protein containing a Gal4 DNA-binding domain in yeast Y190 (19Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5250) Google Scholar). A human lymphocyte MATCHMAKER cDNA library (CLONTECH) was used for yeast two-hybrid interaction with hsRPA3. The transformation and selection procedures were performed according to the CLONTECH manual with slight modifications. The library plasmids harboring RUVBL1 cDNA were extracted from the screened yeast and sequenced. RUVBL1 cDNA sequence has been deposited in the GenBankTM(accession number AF070735). An ∼4.8-kb human genomic clone containing RUVBL1 was identified by screening a human placenta genomic library (CLONTECH) using the RUVBL1 cDNA as probe. The genomic clone was labeled with digoxigenin-11-dUTP as described (20Zhao Y. Bjorbaek C. Weremowicz S. Morton C.C. Moller D.E. Mol. Cell. Biol. 1995; 15: 4353-4363Crossref PubMed Scopus (117) Google Scholar). Hybridization of metaphase chromosome preparations from peripheral blood lymphocytes obtained from normal human males was performed with the RUVBL1 gene at 15 μg/ml in Hybrisol VI according to a previously described method (21Ney P.A. Andrews N.C. Jane S.M. Safer B. Purucker N.E. Weremowicz S. Morton C.C. Goff S.C. Orkin S.H. Nienhuis A.W. Mol. Cell. Biol. 1993; 13: 5604-5612Crossref PubMed Scopus (162) Google Scholar). Human WI38 fibroblasts, 293T transformed embryonic kidney cells, or HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Anti-RUVBL1 antiserum was raised in a rabbit using a recombinant His6-tagged fragment of RUVBL1 containing amino acids 61–456 created by cloning the fragment of RUVBL1 cDNA into the XhoI site of pRSETC (Invitrogen). The antibody was further immunopurified from the antiserum using purified RUVBL1 as antigen. Immunoblotting was performed according to standard protocols. 293T cells were labeled with [35S]methionine for 6 h in methionine-free Dulbecco's modified Eagle's medium following a 4-h starvation and lysed in RIPA buffer (150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 50 mmTris-HCl, pH 8.0, 1 mm DTT, and 0.1 mm PMSF). The lysate was precleared with preimmune serum bound to protein A-Sepharose for 1 h followed by a 1-h incubation with anti-RUVBL1 antiserum in the above lysis buffer. The precipitated complex was loaded on a 12% SDS-PAGE gel, and detected by autoradiography. To ensure that co-immunoprecipitating proteins were not a result of cross-reacting antibody, interactions were disrupted by lysing cells in 1% SDS at 100 °C. Samples were then diluted to RIPA buffer conditions and immunoprecipitated with the same antibodies. Total RNA was extracted from HeLa cells as described (22Chomczynski P. Sacchi N. Anal. Biochem. 1997; 162: 156-159Crossref Scopus (63232) Google Scholar). 10 μg of RNA/lane was separated on a formaldehyde-agarose gel and blotted to a nylon membrane. The blot was hybridized at 42 °C with a fragment of RUVBL1 cDNA encoding amino acids 61–456. The membranes were also hybridized with a 1.3-kbHindIII-PstI cDNA fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cDNA fragments of 1.7 and 1.4 kb containing the entire open reading frames of cyclins B and E, excised out of RcCyclin B and RcCyclin E plasmids withHindIII and XbaI (23Hinds P.W. Millnacht S. Dulic V. Arnold A. Reed S.I. Weinberg R.A. Cell. 1992; 70: 993-1006Abstract Full Text PDF PubMed Scopus (876) Google Scholar). Full-length RUVBL1 coding sequence was cloned intoBamHI/XhoI sites of pFastBac 1 (Life Technologies, Inc.) and transposed into a bacmid following the transformation of DH10 Bac (Life Technologies, Inc.). Then baculovirus bearing RUVBL1 was harvested from Sf9 insect cells transfected with the bacmid and employed to infect High 5 insect cells in Grace's insect medium supplemented with 10% heat-inactivated fetal bovine serum. The infected High 5 cells were harvested and lysed in lysis buffer (50 mm Tris acetate, pH 8.0, 150 mmKOAc, 1 mm EDTA, 10% glycerol, 1 mm DTT, 0.5% Nonidet P-40, and 0.1 mm PMSF). The lysate was first passed through phosphocellulose column equilibrated with TEGD buffer (20 mm Tris acetate, pH 7.7, 1 mm EDTA, 10% glycerol, 1 mm DTT, and 1 mm PMSF). The flow-through (fraction 1) was directly loaded onto a Q Sepharose column equilibrated with 50 mm KOAc in TEGD buffer and eluted with a 50–500 mm KOAc gradient in TEGD buffer. RUVBL1 was in the 200–350 mm KOAc fraction. Following overnight dialysis in TEGD buffer plus 50 mm KOAc, the above fraction containing RUVBL1 (fraction 2) was loaded onto a Mono Q fast protein liquid chromatography column equilibrated with 50 mm KOAc in TEGD buffer and eluted with a 50–350 mm KOAc gradient in TEGD buffer. RUVBL1 was eluted with ∼260 mm KOAc and precipitated with 50% saturated ammonium sulfate for 1 h. The precipitate was dissolved in RUVBL1 storage buffer (20 mmTris acetate, pH 7.7, 50 mm KOAc, 10% glycerol, 0.02 mm EDTA, 1 mm DTT, and 1 mm PMSF), dialyzed in the same buffer at 4 °C, and stored at −70 °C. RUVBL1 was followed in the above different steps by SDS-PAGE of fractions and Western blot with αRUVBL1 antibodies. The RUVBL1 protein purified over these steps was at least 95% pure. Two assays, a TLC assay and a coupled spectrophotometric assay, have been used to measure ATPase activity of bacterial RuvB. They are suitable for measuring ATP hydrolysis rates at ATP concentrations below and above 125 μm, respectively. In the TLC assay (24Marrione P.E. Cox M.M. Biochemistry. 1995; 34: 9809-9818Crossref PubMed Scopus (50) Google Scholar, 25Mitchell A.H. West S.C. J. Biol. Chem. 1996; 271: 19205-19497Google Scholar), reactions were carried out at 37 °C in the absence or presence of various DNAs including single-stranded or double-stranded linear DNAs, circular plasmid or phage DNAs, and synthetic Holliday junction DNAs at 20–200 μm(nucleotides). The reaction mixtures contained 20 mmTris-HCl at pH 6.8–8.0, 1–32 mm MgCl2, 1 mm DTT, 100 μg/ml bovine serum albumin, 25–1300 μm ATP, 40 μCi/ml [α-32P]ATP, 0–0.5 μm hsRPA (18Lin Y.-L. Chen C. Keshav K.F. Winchester E. Dutta A. J. Biol. Chem. 1996; 271: 17190-17198Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and 0.6–4.0 μm purified RUVBL1. The reactions were stopped by addition of EDTA to 40 mm. Aliquots (1 μl) of reaction were spotted at various time points (1–60 min) onto polyethyleneimine-cellulose TLC plates, which were developed in 1 m formic acid/0.5 mLiCl. Hydrolysis of [α-32P]ATP into [α-32P]ADP was determined by autoradiograhpy. The coupled spectrophotometric assay in which ATP hydrolysis was coupled with oxidation of NADH (24Marrione P.E. Cox M.M. Biochemistry. 1995; 34: 9809-9818Crossref PubMed Scopus (50) Google Scholar) employed pyruvate kinase and lactate dehydrogenase as an ATP regeneration system in addition to the reaction components supplemented in TLC assay. Because the oxidation of NADH can be detected at 380 nm by spectrophotometer, [α-32P]ATP was omitted from the reaction mixture. An NADH extinction coefficient of ε380 = 1.21 mm−1 cm−1 was used to calculate the rate of ATP hydrolysis. Two assays using synthetic Holliday junctions and primer/template duplexes as substrates, respectively, were employed to measure branch migration and helicase activity of RUVBL1. Synthetic Holliday junctions were prepared essentially as described previously (26Hiom K. West S.C. Cell. 1995; 80: 787Abstract Full Text PDF PubMed Scopus (62) Google Scholar). The asymmetric Holliday junction was constructed from four oligonucleotides (oligos 1–4) with 88 or 89 bases. Oligo-1 was 5′-32P-labeled prior to annealing using T4 polynucleotide kinase and [γ-32P]ATP. Annealed junctions were purified by gel electrophoresis. The partial duplex markers used in the experiment shown in Fig. 4 B were prepared by annealing 200 ng of32P-labeled oligo-1 with excess oligo-2 or -4. To determine the activity of RUVBL1, the reaction mixture (20 μl) contained ∼2.5 ng of 32P-labeled synthetic Holliday junction DNA in 20 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol, 100 μg/ml bovine serum albumin, 1 mm ATP, and 0.6–4.0 μm RUVBL1 (26Hiom K. West S.C. Cell. 1995; 80: 787Abstract Full Text PDF PubMed Scopus (62) Google Scholar). Reactions lasted for 15–60 min at 37 °C and were stopped and deproteinized by the addition of 2 μl of 10× stop buffer to a final concentration of 20 mm Tris-HCl, pH 7.5, 25 mmEDTA, 0.5% SDS, and 2 mg ml−1 proteinase K. The samples were analyzed using a 6% polyacrylamide gel with a Tris borate buffer system. 32P-Labeled DNAs were detected by autoradiography. For simple helicase assays two primer/template duplexes were prepared in the same manner as the synthetic Holliday junctions. Two template oligonucleotides, 5′ to 3′ template and 3′ to 5′ template were used with their 3′ and 5′ ends, respectively, annealed to the32P-labeled primer. The DNA sequences for these oligonucleotides are the followings: primer, 5′-TGGTATGGTGAGCACTGCAGCCAGGATCAT-3′; 5′ to 3′ template, 5′-TCTCCCTATAGTGAGTCGTATTTTTGATCCTGGCTGCAGTGCTCACCATACCA-3′; 3′ to 5′ template, 5′-ATGATCCTGGCTGCAGTGCTCACCTTACCTTCTCCCTATACTGAGTCGTATTT-3′. Reaction mixtures contained 10 mm Tris-HCl, pH 8.0, 6 mm MgCl2, 1 mm DTT, 2 mm ATP, 2 mm dATP or GTP or dNTP or NTP, 400 μg/ml bovine serum albumin, 2.5 nm of the annealed duplexes, and 1 μm RUVBL1. Following 2 h of incubation at 30 °C, the reactions were stopped by addition of stop buffer to a final concentration of 0.33% SDS, 17 mm EDTA, 14% glycerol, and 0.01% bromphenol blue. The samples were analyzed on an 18% polyacrylamide gel with a Tris borate buffer system.32P-Labeled DNAs were detected by autoradiography. pol II holoenzyme was purified from HeLa whole cell extracts through Bio-Rex 70 column, sucrose step gradient, and nickel nitrilotriacetate as described previously (14Scully R. Anderson S.F. Chao D.M. Wei W. Ye L. Young R.A. Livingston D.M. Parvin J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5605-5610Crossref PubMed Scopus (425) Google Scholar, 27Neish A.S. Anderson S.F. Schlegel B.P. Wei W. Parvin J.D. Nucleic Acids Res. 1998; 26: 847-853Crossref PubMed Scopus (145) Google Scholar). Pull-down of RNA polymerase holoenzyme from cell extracts by GST-CBP (containing amino acid residues 1805–1890 of CBP fused to glutathione S-transferase) or GST-BRCA1 (containing amino acid residues 1560–1863 of the familial breast cancer susceptibility gene product, BRCA1) has also been described in detail (27Neish A.S. Anderson S.F. Schlegel B.P. Wei W. Parvin J.D. Nucleic Acids Res. 1998; 26: 847-853Crossref PubMed Scopus (145) Google Scholar, 28Anderson S.F. Schlegel B.P. Nakajima T. Wolpin E.S. Parvin J.D. Nat. Genet. 1998; 19: 254-256Crossref PubMed Scopus (342) Google Scholar). scRUVBL1 was amplified from yeast genomic DNA using polymerase chain reaction primer 1 (5′-CATGCCATGGTCGCTATCAGTGAAGTCA-3′; the ATG corresponding to the initiator methionine of scRUVBL1, GenBankTM accession number S52968) and primer 2 (5′-GGGGGATCCTTACAAATAATTTGCGGAAGTT-3′; the TTA is antisense to the termination codon TAA of the scRUVBL1 sequence). The 1.4-kb product containing the entire open reading frame of scRUVBL1 was blunted at the NcoI site (by polymerase fill-in reaction) and inserted into pKS+ (Stratagene) between theEcoRV and BamHI sites in the polylinker. To knockout scRUVBL1, pKS+-scRUVBL1 was digested withBglII and EcoRV, removing a 420-bp internal segment of the scRUVBL1 gene. This internal segment was replaced by a 1.2-kb HindIII fragment carrying the URA3 gene such that it was flanked by 630 bp of the 5′ and 330 bp of the 3′ end of scRUVBL1. The entire scRUVBL1-URA3 cassette was removed from the plasmid withSalI and BamHI and transformed into the diploidS. cerevisiae strain YSB455 (MATa/MATα ura3–52/ura3–52 leu2Δ1/leu2Δ1 trp1Δ63/trp1Δ63 his3Δ200/his3Δ200 lys2Δ202/lys2Δ202). Several URA+ transformants were selected. Restriction digest and Southern analysis of genomic DNA identified several colonies with successful deletion of one copy of the scRUVBL1 gene. Sporulation and tetrad dissection was conducted for two independently derived yeast strains with a deletion of scRUVBL1 according to standard protocols. To rescue the above yeast strains bearing a heterozygous deletion of scRUVBL1, a cDNA fragment containing scRUVBL1 open reading frame was inserted into an ectopic expression vector Yep51 (18Lin Y.-L. Chen C. Keshav K.F. Winchester E. Dutta A. J. Biol. Chem. 1996; 271: 17190-17198Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) betweenSalI and BamHI sites. Following transformation of the strains with Yep51-scRUVBL1, the tetrads were sporulated and dissected. The strains were also transformed with Yep51 or Yep51-RUVBL1 instead of Yep51-scRUVBL1. To confirm the results of dissection, each haploid was tested by patch-mating with tester strains MAT amet and MAT α met, respectively. The yeast two-hybrid system is a sensitive in vivo method for identifying genes encoding proteins that interact with a protein of interest. Using hsRPA3 (the 14-kDa subunit of hsRPA) as a bait in the yeast two-hybrid system to screen a human cDNA library, we identified several cDNAs that encode potential hsRPA3-interacting proteins including one for RPA1 (the 70-kDa subunit of hsRPA) and a 1.8-kb novel cDNA. The latter encodes a protein of 456 amino acids (50 kDa), referred to as RUVBL1, and is a human homolog of the recently identified rat TBP-interacting protein (TIP49) (29Kanemaki M. Makino Y. Yoshida T. Kishimoto T. Koga A. Yamamoto K. Yamamoto M. Moncollin V. Egly J.-M. Muramatsu M. Tamura T. Biochem. Biophys. Res. Commun. 1997; 235: 64-68Crossref PubMed Scopus (99) Google Scholar). Amino acid sequences of RUVBL1 and TIP49 proteins are identical except that Ile291 in RUVBL1 is replaced by Val in TIP49. As reported previously (29Kanemaki M. Makino Y. Yoshida T. Kishimoto T. Koga A. Yamamoto K. Yamamoto M. Moncollin V. Egly J.-M. Muramatsu M. Tamura T. Biochem. Biophys. Res. Commun. 1997; 235: 64-68Crossref PubMed Scopus (99) Google Scholar), TIP49 shares high homology with RuvB proteins from different bacteria including Thermus aquaticus thermophilus(Ref. 30Tong J. Wetmur J.G. J. Bacteriol. 1996; 178: 2695-2700Crossref PubMed Scopus (21) Google Scholar; GenBankTM accession number U38840),Thermotoga maritima (30Tong J. Wetmur J.G. J. Bacteriol. 1996; 178: 2695-2700Crossref PubMed Scopus (21) Google Scholar), Mycobacterium leprae(GenBankTM accession number U00011) and Borrelia burgdorferi (GenBankTM accession number Y08885). As shown in Fig. 1, two regions of RUVBL1 (amino acids 26–88 and amino acids 277–425) are homologous to the RuvB sequence of T. thermophilus (30Tong J. Wetmur J.G. J. Bacteriol. 1996; 178: 2695-2700Crossref PubMed Scopus (21) Google Scholar). T. thermophilus RuvB consists of 324 amino acids, and its amino acids 1–226 were aligned with the two regions of RUVBL1 in Fig. 1(A and B). The two homologous regions between the two proteins are 25 and 38% identical, respectively, and 46 and 54% similar, respectively. The regions of homology contain Walker A and B motifs but are not restricted to just those motifs. Walker A (Gx4GKT) and B (4 hydrophobic-DExH/N) motifs are involved in ATP binding and/or ATP hydrolysis of DNA/RNA helicases (31Gorbalenya A.E. Koonin E.V. Donchenko A.P. Blinov V.M. Nucleic Acids Res. 1989; 12: 4713-4730Crossref Scopus (830) Google Scholar, 32Koonin E.V. Nucleic Acids Res. 1993; 11: 2541-2547Crossref Scopus (341) Google Scholar). RUVBL1 contains an insertion of approximately 190 amino acids between the two regions. In a recent paper the bacterial RuvB protein was suggested to be structurally similar to subunits of RF-C and other clamp loader protein complexes associated with replicative DNA polymerases from multiple species (33Guenther B. Onrust R. Sali A. O'Donnell M. Kuriyan J. Cell. 1997; 91: 335-345Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). In support of this, we note a moderate sequence homology between RUVBL1, RuvB, and DNA polymerase III γ and τ subunits (GenBankTM accession number g580914) of Bacillus subtilis (Fig. 1 B). Interestingly, like RUVBL1, the subunits of clamp loader protein complexes have insertions of different sizes between the regions containing the Walker A and B motifs (Ref. 33Guenther B. Onrust R. Sali A. O'Donnell M. Kuriyan J. Cell. 1997; 91: 335-345Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholarand Fig. 1 B). Taken together, we suggest that RUVBL1 is structurally a part of the RuvB/clamp loader subunit family of proteins. Two putative yeast genes were identified in the S. cerevisiae genome encoding proteins scRUVBL1 and scRUVBL2 with 70 and 42% identity to RUVBL1, respectively. They are highly homologous to RUVBL1 over both the regions containing the Walker A and B motifs and over the third inserted region between the two motifs (Fig. 1,A and B). Because protein complexes involved in DNA repair and replication are remarkably well conserved in sequence and subunit composition between mammals and yeast, we anticipate that there are likely to be at least two related RUVBL proteins in humans. The human gene identified is more similar to scRUVBL1 than scRUVBL2 leading us to tentatively identify it as RUVBL1. The human RUVBL1 gene was mapped to chromosome 3 in band q21 (3q21) using fluorescence in situ hybridization. Map position was determined by visual inspection of the fluorescent hybridization signals on 4,6-diamidino-2-phenylindole-dihydrochloride-stained metaphase chromosomes. In 18 of 20 metaphase preparations analyzed, hybridization signal was found to be present on the long arm of chromosome 3 in band q21; in 12 metaphase spreads both copies of chromosome 3 were labeled; and in 6 metaphase spreads signal was detected on one chromosome 3. Because Rad51 mRNA is regulated during the cell cycle (6Flygare J. Benson F. Hellgren D. Biochim. Biophys. Acta. 1996; 1312: 231-236Crossref PubMed Scopus (68) Google Scholar, 7Yamamoto A. Taki T. Yagi H. Habu T. Yoshida K. Yoshimura Y. Yamamoto K. Matsushiro A. Nishimune Y. Morita T. Mol. Gen. Genet. 1996; 251: 1-12Crossref PubMed Google Scholar), it was interesting to determine whether the expression of RUVBL1 is similarly regulated. RUVBL1 mRNA levels were examined by Northern blot analysis of RNA from synchronous HeLa cells released from an M phase block by nocodazole (Fig. 2). The progressive decrease in cyclin B and increase in cyclin E mRNA levels indicates that the cells passed through G1 and S synchronously. However, RUVBL1 mRNA was detected at a constant level during the cell cycle in comparison with the GAPDH control. Thus, unlike Rad51, which shows increased expression in S phase (6Flygare J. Benson F. Hellgren D. Biochim. Biophys. Acta. 1996; 1312: 231-236Crossref PubMed Scopus (68) Google Scholar, 7Yamamoto A. Taki T. Yagi H. Habu T. Yoshida K. Yoshimura Y. Yamamoto K. Matsushiro A. Nishimune Y. Morita T. Mol. Gen. Genet. 1996; 251: 1-12Crossref PubMed Google Scholar), RUVBL1 mRNA is not cell cycle-regulated. Antiserum against RUVBL1 was raised from a rabbit immunized with the His-tagged fragment of RUVBL1 (amino acids 61–456) overexpressed in Escherichia coli. Western blot with this antiserum detected a 50-kDa protein of the expected size in the following human cell lines: 293T (embryonic kidney cells transformed with adenovirus and SV40 T antigen), MCF7 (breast cancer cells), HeLa (cervical cancer cells), and WI38 (primary fibroblasts) (Fig. 3 A, lanes 1 and 2 and data not shown). Because bacterial RuvB alone promotes branch migration and hydrolyzes ATP, we asked if the same was true for RUVBL1. RUVBL1 protein was overexpressed in High 5 insect cells infected with a recombinant baculovirus. In comparison with uninfected cells, a protein of 50 kDa matching the predicted size of intact RUVBL1 and three smaller breakdown products were detected in the infected cells by immunoblotting with anti-RUVBL1 antibody (Fig. 3 A, lanes 3–6). Intact RUVBL1 was purified by conventional chromatography to >95% purity as shown in Fig. 3 B(lane 2). Unexpectedly, the purified RUVBL1 expressed in the insect baculovirus system did not hydrolyze ATP. Fig. 4 A shows that RUVBL1 did not have any ATPase activity detected with TLC assay in the presence of 25 μm ATP. The results were still negative in the presence of higher concentrations of ATP up to 1.3 mm using both the TLC and the coupled spectrophotometric assays under the different buffer conditions described under “Experimental Procedures” despite the addition of different types of DNA substrates. There was no eukaryotic RUVB protein to be used as a positive control, and the conditions required for a prokaryotic RUVB protein may well be different from those that are optimal for the eukaryotic RUVBL1. Thus, High 5 cell lysate was utilized as a positive control to determine the sensitivity of the assays i" @default.
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• W2023489445 title "An Eukaryotic RuvB-like Protein (RUVBL1) Essential for Growth" @default.
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