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. 1998 Sep 15;17(18):5509–5518. doi: 10.1093/emboj/17.18.5509

An ATP-ADP switch in MuB controls progression of the Mu transposition pathway.

M Yamauchi 1, T A Baker 1
PMCID: PMC1170876  PMID: 9736628

Abstract

MuB protein, an ATP-dependent DNA-binding protein, collaborates with Mu transposase to promote efficient transposition. MuB binds target DNA, delivers this target DNA segment to transposase and activates transposase's catalytic functions. Using ATP-bound, ADP-bound and ATPase-defective MuB proteins we investigated how nucleotide binding and hydrolysis control the activities of MuB protein, important for transposition. We found that both MuB-ADP and MuB-ATP stimulate transposase, whereas only MuB-ATP binds with high affinity to DNA. Four different ATPase-defective MuB mutants fail to activate the normal transposition pathway, further indicating that ATP plays critical regulatory roles during transposition. These mutant proteins fall into two classes: class I mutants are defective in target DNA binding, whereas class II mutants bind target DNA, deliver it to transposase, but fail to promote recombination with this DNA. Based on these studies, we propose that the switch from the ATP- to ADP-bound form allows MuB to release the target DNA while maintaining its stimulatory interaction with transposase. Thus, ATP-hydrolysis by MuB appears to function as a molecular switch controlling how target DNA is delivered to the core transposition machinery.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adzuma K., Mizuuchi K. Interaction of proteins located at a distance along DNA: mechanism of target immunity in the Mu DNA strand-transfer reaction. Cell. 1989 Apr 7;57(1):41–47. doi: 10.1016/0092-8674(89)90170-0. [DOI] [PubMed] [Google Scholar]
  2. Adzuma K., Mizuuchi K. Steady-state kinetic analysis of ATP hydrolysis by the B protein of bacteriophage mu. Involvement of protein oligomerization in the ATPase cycle. J Biol Chem. 1991 Apr 5;266(10):6159–6167. [PubMed] [Google Scholar]
  3. Adzuma K., Mizuuchi K. Target immunity of Mu transposition reflects a differential distribution of Mu B protein. Cell. 1988 Apr 22;53(2):257–266. doi: 10.1016/0092-8674(88)90387-x. [DOI] [PubMed] [Google Scholar]
  4. Andrake M. D., Skalka A. M. Retroviral integrase, putting the pieces together. J Biol Chem. 1996 Aug 16;271(33):19633–19636. doi: 10.1074/jbc.271.33.19633. [DOI] [PubMed] [Google Scholar]
  5. Baker T. A., Kremenstova E., Luo L. Complete transposition requires four active monomers in the mu transposase tetramer. Genes Dev. 1994 Oct 15;8(20):2416–2428. doi: 10.1101/gad.8.20.2416. [DOI] [PubMed] [Google Scholar]
  6. Baker T. A., Mizuuchi K. DNA-promoted assembly of the active tetramer of the Mu transposase. Genes Dev. 1992 Nov;6(11):2221–2232. doi: 10.1101/gad.6.11.2221. [DOI] [PubMed] [Google Scholar]
  7. Baker T. A., Mizuuchi M., Savilahti H., Mizuuchi K. Division of labor among monomers within the Mu transposase tetramer. Cell. 1993 Aug 27;74(4):723–733. doi: 10.1016/0092-8674(93)90519-v. [DOI] [PubMed] [Google Scholar]
  8. Baker T. A., Mizuuchi M., Savilahti H., Mizuuchi K. Division of labor among monomers within the Mu transposase tetramer. Cell. 1993 Aug 27;74(4):723–733. doi: 10.1016/0092-8674(93)90519-v. [DOI] [PubMed] [Google Scholar]
  9. Blum S., Schmid S. R., Pause A., Buser P., Linder P., Sonenberg N., Trachsel H. ATP hydrolysis by initiation factor 4A is required for translation initiation in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7664–7668. doi: 10.1073/pnas.89.16.7664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  11. Craig N. L. Target site selection in transposition. Annu Rev Biochem. 1997;66:437–474. doi: 10.1146/annurev.biochem.66.1.437. [DOI] [PubMed] [Google Scholar]
  12. Craig N. L. Unity in transposition reactions. Science. 1995 Oct 13;270(5234):253–254. doi: 10.1126/science.270.5234.253. [DOI] [PubMed] [Google Scholar]
  13. Craig N. L. V(D)J recombination and transposition: closer than expected. Science. 1996 Mar 15;271(5255):1512–1512. doi: 10.1126/science.271.5255.1512. [DOI] [PubMed] [Google Scholar]
  14. Craigie R., Arndt-Jovin D. J., Mizuuchi K. A defined system for the DNA strand-transfer reaction at the initiation of bacteriophage Mu transposition: protein and DNA substrate requirements. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7570–7574. doi: 10.1073/pnas.82.22.7570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Craigie R., Mizuuchi K. Cloning of the A gene of bacteriophage Mu and purification of its product, the Mu transposase. J Biol Chem. 1985 Feb 10;260(3):1832–1835. [PubMed] [Google Scholar]
  16. Craigie R., Mizuuchi K. Transposition of Mu DNA: joining of Mu to target DNA can be uncoupled from cleavage at the ends of Mu. Cell. 1987 Nov 6;51(3):493–501. doi: 10.1016/0092-8674(87)90645-3. [DOI] [PubMed] [Google Scholar]
  17. Darzins A., Kent N. E., Buckwalter M. S., Casadaban M. J. Bacteriophage Mu sites required for transposition immunity. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6826–6830. doi: 10.1073/pnas.85.18.6826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gradia S., Acharya S., Fishel R. The human mismatch recognition complex hMSH2-hMSH6 functions as a novel molecular switch. Cell. 1997 Dec 26;91(7):995–1005. doi: 10.1016/s0092-8674(00)80490-0. [DOI] [PubMed] [Google Scholar]
  19. Jurnak F. Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. Science. 1985 Oct 4;230(4721):32–36. doi: 10.1126/science.3898365. [DOI] [PubMed] [Google Scholar]
  20. Klemm R. D., Austin R. J., Bell S. P. Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex. Cell. 1997 Feb 21;88(4):493–502. doi: 10.1016/s0092-8674(00)81889-9. [DOI] [PubMed] [Google Scholar]
  21. Kruklitis R., Welty D. J., Nakai H. ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis. EMBO J. 1996 Feb 15;15(4):935–944. [PMC free article] [PubMed] [Google Scholar]
  22. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Laurent B. C., Treich I., Carlson M. The yeast SNF2/SWI2 protein has DNA-stimulated ATPase activity required for transcriptional activation. Genes Dev. 1993 Apr;7(4):583–591. doi: 10.1101/gad.7.4.583. [DOI] [PubMed] [Google Scholar]
  24. Lavoie B. D., Chan B. S., Allison R. G., Chaconas G. Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome. EMBO J. 1991 Oct;10(10):3051–3059. doi: 10.1002/j.1460-2075.1991.tb07856.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Leung P. C., Harshey R. M. Two mutations of phage mu transposase that affect strand transfer or interactions with B protein lie in distinct polypeptide domains. J Mol Biol. 1991 May 20;219(2):189–199. doi: 10.1016/0022-2836(91)90561-j. [DOI] [PubMed] [Google Scholar]
  26. Levchenko I., Luo L., Baker T. A. Disassembly of the Mu transposase tetramer by the ClpX chaperone. Genes Dev. 1995 Oct 1;9(19):2399–2408. doi: 10.1101/gad.9.19.2399. [DOI] [PubMed] [Google Scholar]
  27. Levchenko I., Yamauchi M., Baker T. A. ClpX and MuB interact with overlapping regions of Mu transposase: implications for control of the transposition pathway. Genes Dev. 1997 Jun 15;11(12):1561–1572. doi: 10.1101/gad.11.12.1561. [DOI] [PubMed] [Google Scholar]
  28. Lim W. A., Sauer R. T., Lander A. D. Analysis of DNA-protein interactions by affinity coelectrophoresis. Methods Enzymol. 1991;208:196–210. doi: 10.1016/0076-6879(91)08014-9. [DOI] [PubMed] [Google Scholar]
  29. Maxwell A., Craigie R., Mizuuchi K. B protein of bacteriophage mu is an ATPase that preferentially stimulates intermolecular DNA strand transfer. Proc Natl Acad Sci U S A. 1987 Feb;84(3):699–703. doi: 10.1073/pnas.84.3.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Miller J. L., Anderson S. K., Fujita D. J., Chaconas G., Baldwin D. L., Harshey R. M. The nucleotide sequence of the B gene of bacteriophage Mu. Nucleic Acids Res. 1984 Nov 26;12(22):8627–8638. doi: 10.1093/nar/12.22.8627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Millner A., Chaconas G. Disruption of target DNA binding in Mu DNA transposition by alteration of position 99 in the Mu B protein. J Mol Biol. 1998 Jan 16;275(2):233–243. doi: 10.1006/jmbi.1997.1446. [DOI] [PubMed] [Google Scholar]
  32. Mizuuchi K. Polynucleotidyl transfer reactions in transpositional DNA recombination. J Biol Chem. 1992 Oct 25;267(30):21273–21276. [PubMed] [Google Scholar]
  33. Mizuuchi K. Transpositional recombination: mechanistic insights from studies of mu and other elements. Annu Rev Biochem. 1992;61:1011–1051. doi: 10.1146/annurev.bi.61.070192.005051. [DOI] [PubMed] [Google Scholar]
  34. Mizuuchi M., Baker T. A., Mizuuchi K. Assembly of phage Mu transpososomes: cooperative transitions assisted by protein and DNA scaffolds. Cell. 1995 Nov 3;83(3):375–385. doi: 10.1016/0092-8674(95)90115-9. [DOI] [PubMed] [Google Scholar]
  35. Mizuuchi M., Baker T. A., Mizuuchi K. Assembly of the active form of the transposase-Mu DNA complex: a critical control point in Mu transposition. Cell. 1992 Jul 24;70(2):303–311. doi: 10.1016/0092-8674(92)90104-k. [DOI] [PubMed] [Google Scholar]
  36. Mizuuchi M., Mizuuchi K. Target site selection in transposition of phage Mu. Cold Spring Harb Symp Quant Biol. 1993;58:515–523. doi: 10.1101/sqb.1993.058.01.058. [DOI] [PubMed] [Google Scholar]
  37. Müller C. W., Schulz G. E. Structure of the complex between adenylate kinase from Escherichia coli and the inhibitor Ap5A refined at 1.9 A resolution. A model for a catalytic transition state. J Mol Biol. 1992 Mar 5;224(1):159–177. doi: 10.1016/0022-2836(92)90582-5. [DOI] [PubMed] [Google Scholar]
  38. Naigamwalla D. Z., Chaconas G. A new set of Mu DNA transposition intermediates: alternate pathways of target capture preceding strand transfer. EMBO J. 1997 Sep 1;16(17):5227–5234. doi: 10.1093/emboj/16.17.5227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nakai H., Kruklitis R. Disassembly of the bacteriophage Mu transposase for the initiation of Mu DNA replication. J Biol Chem. 1995 Aug 18;270(33):19591–19598. doi: 10.1074/jbc.270.33.19591. [DOI] [PubMed] [Google Scholar]
  40. Pai E. F., Krengel U., Petsko G. A., Goody R. S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990 Aug;9(8):2351–2359. doi: 10.1002/j.1460-2075.1990.tb07409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pause A., Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 1992 Jul;11(7):2643–2654. doi: 10.1002/j.1460-2075.1992.tb05330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pérez-Martín J., de Lorenzo V. ATP binding to the sigma 54-dependent activator XylR triggers a protein multimerization cycle catalyzed by UAS DNA. Cell. 1996 Jul 26;86(2):331–339. doi: 10.1016/s0092-8674(00)80104-x. [DOI] [PubMed] [Google Scholar]
  43. Reyes O., Beyou A., Mignotte-Vieux C., Richaud F. Mini-Mu transduction: cis-inhibition of the insertion of Mud transposons. Plasmid. 1987 Nov;18(3):183–192. doi: 10.1016/0147-619x(87)90061-8. [DOI] [PubMed] [Google Scholar]
  44. Savilahti H., Rice P. A., Mizuuchi K. The phage Mu transpososome core: DNA requirements for assembly and function. EMBO J. 1995 Oct 2;14(19):4893–4903. doi: 10.1002/j.1460-2075.1995.tb00170.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Story R. M., Steitz T. A. Structure of the recA protein-ADP complex. Nature. 1992 Jan 23;355(6358):374–376. doi: 10.1038/355374a0. [DOI] [PubMed] [Google Scholar]
  46. Surette M. G., Buch S. J., Chaconas G. Transpososomes: stable protein-DNA complexes involved in the in vitro transposition of bacteriophage Mu DNA. Cell. 1987 Apr 24;49(2):253–262. doi: 10.1016/0092-8674(87)90566-6. [DOI] [PubMed] [Google Scholar]
  47. Surette M. G., Chaconas G. Stimulation of the Mu DNA strand cleavage and intramolecular strand transfer reactions by the Mu B protein is independent of stable binding of the Mu B protein to DNA. J Biol Chem. 1991 Sep 15;266(26):17306–17313. [PubMed] [Google Scholar]
  48. Surette M. G., Harkness T., Chaconas G. Stimulation of the Mu A protein-mediated strand cleavage reaction by the Mu B protein, and the requirement of DNA nicking for stable type 1 transpososome formation. In vitro transposition characteristics of mini-Mu plasmids carrying terminal base pair mutations. J Biol Chem. 1991 Feb 15;266(5):3118–3124. [PubMed] [Google Scholar]
  49. Teplow D. B., Nakayama C., Leung P. C., Harshey R. M. Structure-function relationships in the transposition protein B of bacteriophage Mu. J Biol Chem. 1988 Aug 5;263(22):10851–10857. [PubMed] [Google Scholar]
  50. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Watson M. A., Chaconas G. Three-site synapsis during Mu DNA transposition: a critical intermediate preceding engagement of the active site. Cell. 1996 May 3;85(3):435–445. doi: 10.1016/s0092-8674(00)81121-6. [DOI] [PubMed] [Google Scholar]
  52. Welty D. J., Jones J. M., Nakai H. Communication of ClpXP protease hypersensitivity to bacteriophage Mu repressor isoforms. J Mol Biol. 1997 Sep 12;272(1):31–41. doi: 10.1006/jmbi.1997.1193. [DOI] [PubMed] [Google Scholar]
  53. la Cour T. F., Nyborg J., Thirup S., Clark B. F. Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography. EMBO J. 1985 Sep;4(9):2385–2388. doi: 10.1002/j.1460-2075.1985.tb03943.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. van Gent D. C., Mizuuchi K., Gellert M. Similarities between initiation of V(D)J recombination and retroviral integration. Science. 1996 Mar 15;271(5255):1592–1594. doi: 10.1126/science.271.5255.1592. [DOI] [PubMed] [Google Scholar]

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