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W2002180117 abstract "The Escherichia coli RuvB protein is a motor protein that forms a complex with RuvA and promotes branch migration of Holliday junctions during homologous recombination. This study describes the characteristics of two RuvB mutants, I148T and I150T, that do not promote branch migration in the presence of RuvA. These RuvB mutants hydrolyzed ATP and bound duplex DNA with the same efficiency as wild-type RuvB, but the mutants did not form a complex with RuvA and were defective in loading onto junction DNA in a RuvA-assisted manner. A recent crystallographic study revealed that Ile148 and Ile150 are in a unique β-hairpin that protrudes from the AAA+ ATPase domain of RuvB. We propose that this β-hairpin interacts with hydrophobic residues in the mobile third domain of RuvA and that this interaction is vital for the RuvA-assisted loading of RuvB onto Holliday junction DNA. The Escherichia coli RuvB protein is a motor protein that forms a complex with RuvA and promotes branch migration of Holliday junctions during homologous recombination. This study describes the characteristics of two RuvB mutants, I148T and I150T, that do not promote branch migration in the presence of RuvA. These RuvB mutants hydrolyzed ATP and bound duplex DNA with the same efficiency as wild-type RuvB, but the mutants did not form a complex with RuvA and were defective in loading onto junction DNA in a RuvA-assisted manner. A recent crystallographic study revealed that Ile148 and Ile150 are in a unique β-hairpin that protrudes from the AAA+ ATPase domain of RuvB. We propose that this β-hairpin interacts with hydrophobic residues in the mobile third domain of RuvA and that this interaction is vital for the RuvA-assisted loading of RuvB onto Holliday junction DNA. adenosine 5′-O-(thiotriphosphate) Homologous recombination plays important biological roles in regulating genetic diversity and in repairing damaged chromosomes. Homologous recombination involves a series of enzymatic reactions carried out by large multiprotein complexes. One intermediate of homologous recombination is a four-stranded DNA structure called a Holliday junction (1Holliday R. Genet. Res. 1964; 5: 282-304Crossref Scopus (1261) Google Scholar). In bacteria, Holliday junctions are processed at a late stage of recombination into two recombinant duplex DNA molecules by a protein complex that includes RuvA, RuvB, and RuvC (2Shinagawa H. Iwasaki H. Trends Biochem. Sci. 1996; 21: 107-111Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 3West S.C. Annu. Rev. Genet. 1997; 31: 213-244Crossref PubMed Scopus (386) Google Scholar).RuvA and RuvB form a complex that promotes movement of a Holliday junction, a process known as branch migration. Electron microscopic studies have demonstrated that RuvB forms a hexameric ring that encircles duplex DNA (4Stasiak A. Tsaneva I.R. West S.C. Benson C.J., Yu, X. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7618-7622Crossref PubMed Scopus (143) Google Scholar). Two RuvB hexameric rings flank two RuvA tetramers that sandwich the Holliday junction (5Yu X. West S.C. Egelman E.H. J. Mol. Biol. 1997; 266: 217-222Crossref PubMed Scopus (67) Google Scholar). This structure suggests that homologous (homeologous) DNA duplexes are unwound and rewound during branch migration while they pass through the RuvB rings via RuvA tetramers; this process leads to the formation of heteroduplex DNA.The RuvA tetramer is a junction-specific binding protein that interacts directly with RuvB and loads RuvB onto Holliday junctions. The RuvA monomer consists of three domains: domains I and II are involved in tetramer formation and Holliday junction recognition, respectively (6Rafferty J.B. Sedelnikova S.E. Hargreaves D. Artymiuk P.J. Baker P.J. Sharples G.J. Mahdi A.A. Lloyd R.G. Rice D.W. Science. 1996; 274: 415-421Crossref PubMed Scopus (150) Google Scholar, 7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 8Ariyoshi M. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8257-8262Crossref PubMed Scopus (119) Google Scholar). Domain III is highly mobile, is connected with domain II via a flexible loop, and is involved in a specific interaction with RuvB (7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar,9Nishino T. Iwasaki H. Kataoka M. Ariyoshi M. Fujita T. Shinagawa H. Morikawa K. J. Mol. Biol. 2000; 298: 407-416Crossref PubMed Scopus (27) Google Scholar).The RuvB hexamer is a motor that drives branch migration using energy derived from ATP hydrolysis (10Iwasaki H. Takahagi M. Nakata A. Shinagawa H. Genes Dev. 1992; 6: 2214-2220Crossref PubMed Scopus (148) Google Scholar, 11Tsaneva I.R. Müller B. West S.C. Cell. 1992; 69: 1171-1180Abstract Full Text PDF PubMed Scopus (212) Google Scholar). The RuvB ATPase is synergistically stimulated by RuvA and DNA in vitro (12Shiba T. Iwasaki H. Nakata A. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8445-8449Crossref PubMed Scopus (94) Google Scholar). RuvB can be dimeric, hexameric, heptameric, or dodecameric depending on conditions and cofactors such as ATP, Mg2+, and DNA (13Shiba T. Iwasaki H. Nakata A. Shinagawa H. Mol. Gen. Genet. 1993; 237: 395-399Crossref PubMed Scopus (40) Google Scholar, 14Mitchell A.H. West S.C. J. Mol. Biol. 1994; 243: 208-215Crossref PubMed Scopus (63) Google Scholar, 15Miyata T. Yamada K. Iwasaki H. Shinagawa H. Morikawa K. Mayanagi K. J. Struct. Biol. 2000; 131: 83-89Crossref PubMed Scopus (48) Google Scholar). It also interacts with RuvC Holliday junction resolvase (16Eggleston A.K. Mitchell A.H. West S.C. Cell. 1997; 89: 607-617Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar).The RuvB protein is a member of the AAA+ class of ATPases (17Iwasaki H. Han Y.-W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar). The crystal structure of RuvB from Thermus thermophilus HB8 was recently determined (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). This protein has a crescent-like architecture consisting of three consecutive domains. The first two domains have a folding pattern that is well conserved in AAA/AAA+ ATPases and is involved in ATP binding and hydrolysis. However, sequence alignments of AAA+ class proteins show that the amino acid sequence from Leu135 to Leu152 in Escherichia coli RuvB is not conserved in other AAA/AAA+ class proteins such asN-ethylmaleimide-sensitive factor D2. This implies that this unique region is involved in a specific function of RuvB (17Iwasaki H. Han Y.-W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar). This region forms β-hairpin 1, which protrudes from the first domain of RuvB (Fig. 1) (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar).This report describes the properties of two mutant RuvB proteins with mutations in β-hairpin 1, I148T and I150T, which were isolated in a previous study (17Iwasaki H. Han Y.-W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar). The two mutants have a similar phenotype in vivo and are defective in their functional and physical interactions with RuvA protein in vitro. We propose that residues Ile148 and Ile150 in β-hairpin 1 interact with hydrophobic residues in the mobile domain III of RuvA and that this interaction is essential for RuvAB-dependent branch migration.DISCUSSIONThis study examined the properties of RuvB I148T and I150T, which have mutations in β-hairpin 1. DNA stimulated the ATPase activity of the mutant and wild-type RuvB proteins similarly, but RuvA (or RuvA and DNA) stimulated the ATPase activity of the mutants to a much lower extent than it stimulated that of wild-type RuvB (Table I). The RuvB mutants were also deficient in the ability to form a complex with RuvA or a ternary complex with RuvA and a Holliday junction (Figs. 4 and 5). RuvB I148T and I150T bound duplex DNA and formed hexameric rings (Figs.6 and 7) and interacted with RuvC (data not shown) in a manner similar to wild-type RuvB. Thus, the interaction of RuvB I148T and I150T with RuvA, which is required for the elevated ATPase and branch migration activities of the RuvA-RuvB complex, is defective.The data also indicate that RuvB I148T is less severely impaired than RuvB I150T in ternary complex formation, ATP hydrolysis, and branch migration. Complementation analysis also showed that RuvB I148T retained more UV repair activity than RuvB I150T when the proteins were expressed highly in the mutant cells (Fig. 2). These findings further support the idea that the mutants are defective in the interaction with RuvA, and the result of the complementation analysis can be explained by proposing that the decrease in affinity of the mutant proteins can be compensated for by an increase in their concentration.The crystal structure of RuvB from T. thermophilus HB8 was recently determined (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). The RuvB monomer consists of three domains (I, II, and III) that form a crescent-shaped configuration (Fig.1 B). The RuvB-specific region (L135–L152) forms β-hairpin 1, which is composed of the fourth and fifth β-strands. This β-hairpin protrudes from the AAA+ ATPase motif in domain I (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). Leu148 and Leu150 are located in the fifth β-strand (β5). Electron microscopic studies demonstrated that the RuvB hexameric ring includes a large tier and a small tier, and the large tier faces RuvA (4Stasiak A. Tsaneva I.R. West S.C. Benson C.J., Yu, X. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7618-7622Crossref PubMed Scopus (143) Google Scholar, 5Yu X. West S.C. Egelman E.H. J. Mol. Biol. 1997; 266: 217-222Crossref PubMed Scopus (67) Google Scholar, 15Miyata T. Yamada K. Iwasaki H. Shinagawa H. Morikawa K. Mayanagi K. J. Struct. Biol. 2000; 131: 83-89Crossref PubMed Scopus (48) Google Scholar). A tentative model of the hexameric ring based on the crystal structure shows that all six β-hairpin 1 motifs are located on the top of the large tier (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). This is consistent with the idea that β-hairpin 1 is involved in the interface between RuvB and RuvA.It is particularly intriguing that ruvA mutations in hydrophobic residues such as Leu167, Leu170, Tyr172, and Leu199 cause a defect in the RuvA-RuvB interaction. These residues are in mobile domain III of RuvA, which interacts specifically with RuvB (7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Nishino T. Iwasaki H. Kataoka M. Ariyoshi M. Fujita T. Shinagawa H. Morikawa K. J. Mol. Biol. 2000; 298: 407-416Crossref PubMed Scopus (27) Google Scholar). Hydrophobic residues are well conserved in these positions involved in this interaction (7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 17Iwasaki H. Han Y.-W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar). Therefore, the protruding β-hairpin 1 in the AAA+ ATPase domain of RuvB may interact with hydrophobic residues in domain III of RuvA.The mobile domain III of RuvA has also been shown not only to interact physically with RuvB but also to modulate RuvB ATPase activity. This suggests that the signal by RuvA for interaction with DNA may be transduced through the NH2 region (domains I + II) to domain III of RuvA, resulting in continuous cycling of RuvB ATP hydrolysis (7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Nishino T. Iwasaki H. Kataoka M. Ariyoshi M. Fujita T. Shinagawa H. Morikawa K. J. Mol. Biol. 2000; 298: 407-416Crossref PubMed Scopus (27) Google Scholar). Likewise, such a signal may also be transduced through domain III of RuvA to β-hairpin 1 in domain I of RuvB. β-Hairpin 1 of RuvB is situated between the fourth α-helix and the sixth β-sheet (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). It has been proposed that the fourth α-helix is involved in intersubunit interaction, which may couple ATP binding or hydrolysis, and that the sixth β-sheet is involved in sensing the ATP hydrolysis status of its own subunit (17Iwasaki H. Han Y.-W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar, 18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). Therefore, not only is β-hairpin 1 involved in the physical interaction per se with RuvA, but also the interaction with RuvA via β-hairpin 1 may cause a structural change of the ATPase domain of RuvB leading to efficient ATPase cycling. These physical and functional interactions may result in the regulation of RuvB motor activity to drive branch migration of the Holliday junction processively. We propose that β-hairpin 1 interacts with RuvA in a structural and regulatory manner: this interaction may change the structure and activity of the RuvB ATPase domain and drive processive branch migration of Holliday junctions. Homologous recombination plays important biological roles in regulating genetic diversity and in repairing damaged chromosomes. Homologous recombination involves a series of enzymatic reactions carried out by large multiprotein complexes. One intermediate of homologous recombination is a four-stranded DNA structure called a Holliday junction (1Holliday R. Genet. Res. 1964; 5: 282-304Crossref Scopus (1261) Google Scholar). In bacteria, Holliday junctions are processed at a late stage of recombination into two recombinant duplex DNA molecules by a protein complex that includes RuvA, RuvB, and RuvC (2Shinagawa H. Iwasaki H. Trends Biochem. Sci. 1996; 21: 107-111Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 3West S.C. Annu. Rev. Genet. 1997; 31: 213-244Crossref PubMed Scopus (386) Google Scholar). RuvA and RuvB form a complex that promotes movement of a Holliday junction, a process known as branch migration. Electron microscopic studies have demonstrated that RuvB forms a hexameric ring that encircles duplex DNA (4Stasiak A. Tsaneva I.R. West S.C. Benson C.J., Yu, X. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7618-7622Crossref PubMed Scopus (143) Google Scholar). Two RuvB hexameric rings flank two RuvA tetramers that sandwich the Holliday junction (5Yu X. West S.C. Egelman E.H. J. Mol. Biol. 1997; 266: 217-222Crossref PubMed Scopus (67) Google Scholar). This structure suggests that homologous (homeologous) DNA duplexes are unwound and rewound during branch migration while they pass through the RuvB rings via RuvA tetramers; this process leads to the formation of heteroduplex DNA. The RuvA tetramer is a junction-specific binding protein that interacts directly with RuvB and loads RuvB onto Holliday junctions. The RuvA monomer consists of three domains: domains I and II are involved in tetramer formation and Holliday junction recognition, respectively (6Rafferty J.B. Sedelnikova S.E. Hargreaves D. Artymiuk P.J. Baker P.J. Sharples G.J. Mahdi A.A. Lloyd R.G. Rice D.W. Science. 1996; 274: 415-421Crossref PubMed Scopus (150) Google Scholar, 7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 8Ariyoshi M. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8257-8262Crossref PubMed Scopus (119) Google Scholar). Domain III is highly mobile, is connected with domain II via a flexible loop, and is involved in a specific interaction with RuvB (7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar,9Nishino T. Iwasaki H. Kataoka M. Ariyoshi M. Fujita T. Shinagawa H. Morikawa K. J. Mol. Biol. 2000; 298: 407-416Crossref PubMed Scopus (27) Google Scholar). The RuvB hexamer is a motor that drives branch migration using energy derived from ATP hydrolysis (10Iwasaki H. Takahagi M. Nakata A. Shinagawa H. Genes Dev. 1992; 6: 2214-2220Crossref PubMed Scopus (148) Google Scholar, 11Tsaneva I.R. Müller B. West S.C. Cell. 1992; 69: 1171-1180Abstract Full Text PDF PubMed Scopus (212) Google Scholar). The RuvB ATPase is synergistically stimulated by RuvA and DNA in vitro (12Shiba T. Iwasaki H. Nakata A. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8445-8449Crossref PubMed Scopus (94) Google Scholar). RuvB can be dimeric, hexameric, heptameric, or dodecameric depending on conditions and cofactors such as ATP, Mg2+, and DNA (13Shiba T. Iwasaki H. Nakata A. Shinagawa H. Mol. Gen. Genet. 1993; 237: 395-399Crossref PubMed Scopus (40) Google Scholar, 14Mitchell A.H. West S.C. J. Mol. Biol. 1994; 243: 208-215Crossref PubMed Scopus (63) Google Scholar, 15Miyata T. Yamada K. Iwasaki H. Shinagawa H. Morikawa K. Mayanagi K. J. Struct. Biol. 2000; 131: 83-89Crossref PubMed Scopus (48) Google Scholar). It also interacts with RuvC Holliday junction resolvase (16Eggleston A.K. Mitchell A.H. West S.C. Cell. 1997; 89: 607-617Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The RuvB protein is a member of the AAA+ class of ATPases (17Iwasaki H. Han Y.-W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar). The crystal structure of RuvB from Thermus thermophilus HB8 was recently determined (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). This protein has a crescent-like architecture consisting of three consecutive domains. The first two domains have a folding pattern that is well conserved in AAA/AAA+ ATPases and is involved in ATP binding and hydrolysis. However, sequence alignments of AAA+ class proteins show that the amino acid sequence from Leu135 to Leu152 in Escherichia coli RuvB is not conserved in other AAA/AAA+ class proteins such asN-ethylmaleimide-sensitive factor D2. This implies that this unique region is involved in a specific function of RuvB (17Iwasaki H. Han Y.-W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar). This region forms β-hairpin 1, which protrudes from the first domain of RuvB (Fig. 1) (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). This report describes the properties of two mutant RuvB proteins with mutations in β-hairpin 1, I148T and I150T, which were isolated in a previous study (17Iwasaki H. Han Y.-W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar). The two mutants have a similar phenotype in vivo and are defective in their functional and physical interactions with RuvA protein in vitro. We propose that residues Ile148 and Ile150 in β-hairpin 1 interact with hydrophobic residues in the mobile domain III of RuvA and that this interaction is essential for RuvAB-dependent branch migration. DISCUSSIONThis study examined the properties of RuvB I148T and I150T, which have mutations in β-hairpin 1. DNA stimulated the ATPase activity of the mutant and wild-type RuvB proteins similarly, but RuvA (or RuvA and DNA) stimulated the ATPase activity of the mutants to a much lower extent than it stimulated that of wild-type RuvB (Table I). The RuvB mutants were also deficient in the ability to form a complex with RuvA or a ternary complex with RuvA and a Holliday junction (Figs. 4 and 5). RuvB I148T and I150T bound duplex DNA and formed hexameric rings (Figs.6 and 7) and interacted with RuvC (data not shown) in a manner similar to wild-type RuvB. Thus, the interaction of RuvB I148T and I150T with RuvA, which is required for the elevated ATPase and branch migration activities of the RuvA-RuvB complex, is defective.The data also indicate that RuvB I148T is less severely impaired than RuvB I150T in ternary complex formation, ATP hydrolysis, and branch migration. Complementation analysis also showed that RuvB I148T retained more UV repair activity than RuvB I150T when the proteins were expressed highly in the mutant cells (Fig. 2). These findings further support the idea that the mutants are defective in the interaction with RuvA, and the result of the complementation analysis can be explained by proposing that the decrease in affinity of the mutant proteins can be compensated for by an increase in their concentration.The crystal structure of RuvB from T. thermophilus HB8 was recently determined (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). The RuvB monomer consists of three domains (I, II, and III) that form a crescent-shaped configuration (Fig.1 B). The RuvB-specific region (L135–L152) forms β-hairpin 1, which is composed of the fourth and fifth β-strands. This β-hairpin protrudes from the AAA+ ATPase motif in domain I (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). Leu148 and Leu150 are located in the fifth β-strand (β5). Electron microscopic studies demonstrated that the RuvB hexameric ring includes a large tier and a small tier, and the large tier faces RuvA (4Stasiak A. Tsaneva I.R. West S.C. Benson C.J., Yu, X. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7618-7622Crossref PubMed Scopus (143) Google Scholar, 5Yu X. West S.C. Egelman E.H. J. Mol. Biol. 1997; 266: 217-222Crossref PubMed Scopus (67) Google Scholar, 15Miyata T. Yamada K. Iwasaki H. Shinagawa H. Morikawa K. Mayanagi K. J. Struct. Biol. 2000; 131: 83-89Crossref PubMed Scopus (48) Google Scholar). A tentative model of the hexameric ring based on the crystal structure shows that all six β-hairpin 1 motifs are located on the top of the large tier (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). This is consistent with the idea that β-hairpin 1 is involved in the interface between RuvB and RuvA.It is particularly intriguing that ruvA mutations in hydrophobic residues such as Leu167, Leu170, Tyr172, and Leu199 cause a defect in the RuvA-RuvB interaction. These residues are in mobile domain III of RuvA, which interacts specifically with RuvB (7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Nishino T. Iwasaki H. Kataoka M. Ariyoshi M. Fujita T. Shinagawa H. Morikawa K. J. Mol. Biol. 2000; 298: 407-416Crossref PubMed Scopus (27) Google Scholar). Hydrophobic residues are well conserved in these positions involved in this interaction (7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 17Iwasaki H. Han Y.-W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar). Therefore, the protruding β-hairpin 1 in the AAA+ ATPase domain of RuvB may interact with hydrophobic residues in domain III of RuvA.The mobile domain III of RuvA has also been shown not only to interact physically with RuvB but also to modulate RuvB ATPase activity. This suggests that the signal by RuvA for interaction with DNA may be transduced through the NH2 region (domains I + II) to domain III of RuvA, resulting in continuous cycling of RuvB ATP hydrolysis (7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Nishino T. Iwasaki H. Kataoka M. Ariyoshi M. Fujita T. Shinagawa H. Morikawa K. J. Mol. Biol. 2000; 298: 407-416Crossref PubMed Scopus (27) Google Scholar). Likewise, such a signal may also be transduced through domain III of RuvA to β-hairpin 1 in domain I of RuvB. β-Hairpin 1 of RuvB is situated between the fourth α-helix and the sixth β-sheet (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). It has been proposed that the fourth α-helix is involved in intersubunit interaction, which may couple ATP binding or hydrolysis, and that the sixth β-sheet is involved in sensing the ATP hydrolysis status of its own subunit (17Iwasaki H. Han Y.-W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar, 18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). Therefore, not only is β-hairpin 1 involved in the physical interaction per se with RuvA, but also the interaction with RuvA via β-hairpin 1 may cause a structural change of the ATPase domain of RuvB leading to efficient ATPase cycling. These physical and functional interactions may result in the regulation of RuvB motor activity to drive branch migration of the Holliday junction processively. We propose that β-hairpin 1 interacts with RuvA in a structural and regulatory manner: this interaction may change the structure and activity of the RuvB ATPase domain and drive processive branch migration of Holliday junctions. This study examined the properties of RuvB I148T and I150T, which have mutations in β-hairpin 1. DNA stimulated the ATPase activity of the mutant and wild-type RuvB proteins similarly, but RuvA (or RuvA and DNA) stimulated the ATPase activity of the mutants to a much lower extent than it stimulated that of wild-type RuvB (Table I). The RuvB mutants were also deficient in the ability to form a complex with RuvA or a ternary complex with RuvA and a Holliday junction (Figs. 4 and 5). RuvB I148T and I150T bound duplex DNA and formed hexameric rings (Figs.6 and 7) and interacted with RuvC (data not shown) in a manner similar to wild-type RuvB. Thus, the interaction of RuvB I148T and I150T with RuvA, which is required for the elevated ATPase and branch migration activities of the RuvA-RuvB complex, is defective. The data also indicate that RuvB I148T is less severely impaired than RuvB I150T in ternary complex formation, ATP hydrolysis, and branch migration. Complementation analysis also showed that RuvB I148T retained more UV repair activity than RuvB I150T when the proteins were expressed highly in the mutant cells (Fig. 2). These findings further support the idea that the mutants are defective in the interaction with RuvA, and the result of the complementation analysis can be explained by proposing that the decrease in affinity of the mutant proteins can be compensated for by an increase in their concentration. The crystal structure of RuvB from T. thermophilus HB8 was recently determined (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). The RuvB monomer consists of three domains (I, II, and III) that form a crescent-shaped configuration (Fig.1 B). The RuvB-specific region (L135–L152) forms β-hairpin 1, which is composed of the fourth and fifth β-strands. This β-hairpin protrudes from the AAA+ ATPase motif in domain I (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). Leu148 and Leu150 are located in the fifth β-strand (β5). Electron microscopic studies demonstrated that the RuvB hexameric ring includes a large tier and a small tier, and the large tier faces RuvA (4Stasiak A. Tsaneva I.R. West S.C. Benson C.J., Yu, X. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7618-7622Crossref PubMed Scopus (143) Google Scholar, 5Yu X. West S.C. Egelman E.H. J. Mol. Biol. 1997; 266: 217-222Crossref PubMed Scopus (67) Google Scholar, 15Miyata T. Yamada K. Iwasaki H. Shinagawa H. Morikawa K. Mayanagi K. J. Struct. Biol. 2000; 131: 83-89Crossref PubMed Scopus (48) Google Scholar). A tentative model of the hexameric ring based on the crystal structure shows that all six β-hairpin 1 motifs are located on the top of the large tier (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). This is consistent with the idea that β-hairpin 1 is involved in the interface between RuvB and RuvA. It is particularly intriguing that ruvA mutations in hydrophobic residues such as Leu167, Leu170, Tyr172, and Leu199 cause a defect in the RuvA-RuvB interaction. These residues are in mobile domain III of RuvA, which interacts specifically with RuvB (7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Nishino T. Iwasaki H. Kataoka M. Ariyoshi M. Fujita T. Shinagawa H. Morikawa K. J. Mol. Biol. 2000; 298: 407-416Crossref PubMed Scopus (27) Google Scholar). Hydrophobic residues are well conserved in these positions involved in this interaction (7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 17Iwasaki H. Han Y.-W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar). Therefore, the protruding β-hairpin 1 in the AAA+ ATPase domain of RuvB may interact with hydrophobic residues in domain III of RuvA. The mobile domain III of RuvA has also been shown not only to interact physically with RuvB but also to modulate RuvB ATPase activity. This suggests that the signal by RuvA for interaction with DNA may be transduced through the NH2 region (domains I + II) to domain III of RuvA, resulting in continuous cycling of RuvB ATP hydrolysis (7Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Nishino T. Iwasaki H. Kataoka M. Ariyoshi M. Fujita T. Shinagawa H. Morikawa K. J. Mol. Biol. 2000; 298: 407-416Crossref PubMed Scopus (27) Google Scholar). Likewise, such a signal may also be transduced through domain III of RuvA to β-hairpin 1 in domain I of RuvB. β-Hairpin 1 of RuvB is situated between the fourth α-helix and the sixth β-sheet (18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). It has been proposed that the fourth α-helix is involved in intersubunit interaction, which may couple ATP binding or hydrolysis, and that the sixth β-sheet is involved in sensing the ATP hydrolysis status of its own subunit (17Iwasaki H. Han Y.-W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar, 18Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). Therefore, not only is β-hairpin 1 involved in the physical interaction per se with RuvA, but also the interaction with RuvA via β-hairpin 1 may cause a structural change of the ATPase domain of RuvB leading to efficient ATPase cycling. These physical and functional interactions may result in the regulation of RuvB motor activity to drive branch migration of the Holliday junction processively. We propose that β-hairpin 1 interacts with RuvA in a structural and regulatory manner: this interaction may change the structure and activity of the RuvB ATPase domain and drive processive branch migration of Holliday junctions." @default.
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W2002180117 title "A Unique β-Hairpin Protruding from AAA+ATPase Domain of RuvB Motor Protein Is Involved in the Interaction with RuvA DNA Recognition Protein for Branch Migration of Holliday Junctions" @default.
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