EPR &RESONANCE RAMAN OF CHLAMYDOMONAS CHLOROPLAST HEMOGLOBIN (HB) &MUTANTS

Project: Research project

Project Details

Description

DNA transposition involves the integration of a transposable DNA element (transposon) into a new target chromosome. The process is a common source of mutations and genome rearrangements, and is essential to the mechanism of antibiotic resistance and to the function of retroviruses. The bacterial virus Mu utilizes the tetrameric protein MuA transposase both to cleave DNA and to join the two ends of the transposon to the host chromosome. The divalent metal cations magnesium or manganese are required for both DNA cleavage and joining, but details of the metal binding are poorly understood. The study will utilize multifrequency (4-18, 140 GHz) cw and pulsed EPR to address issues regarding metal binding in Mu transposase. Initial X-band cw studies will determine the number and dissociation constants of Mn(II) bound at various stages of protein-DNA assembly and activity (i.e. monomer stage, tetramer formation-DNA binding stage, cleavage and joining stages). These experiments will be followed by multifrequency pulsed EPR studies (ESEEM and ENDOR) to determine structural aspects of the specific metal binding site(s). Site specific mutagenesis at specific amino acid resideues (Asp269, Asp336, Glu392) severely compromises both DNA cleavage and strand transfer activities of MuA, giving indirect evidence3 for a metal bvinding site. ESEEM/ENDOR/High frequency EPR with 2H, 13C, 15N, 17O-labeled Asp and Glu will provide direct evidence of the binding of these residues via measured hyperfine interactions. Again, these measurements can be made at different stages of assembly/activity to determine changes in active site structure. Other divalent metals (Co(II), Cu(II), VO(II)) may be used as complementary probes of the metal binding site(s).
StatusFinished
Effective start/end date10/1/959/30/99

Funding

  • National Center for Research Resources

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  • BIOTECHNOLOGY RESOURCE IN PULSED EPR SPECTROSCOPY

    Peisach, J., Aisen, P., Armstrong, W. H., Ash, D. E., Astroff, N., Avdievich, N., Baker, T. A., Banerjee, R. V., Bender, C., Benkovic, S., Brewer, C. F., Brewer, F., Britt, D., Cammack, R., Caradonna, J., Chan, J., Colaneri, M., Danzinger, N. R., Das, T., Dooley, D., Dyson, H. J., Ettinger, M., Fabry, M. E., Fee, J. A., Frank, N. H. A., Friedman, J. M., Gerfen, G. J., Goutermann, M., Guajardo, G. B., Janson, E., Kamp, N., Kozarich, J., Lee, C. C., Lippai, I., Magliozzo, R. S., Mason, R., McMillin, D., Michael, D., Mildvan, A., Mims, W. B., Narasimhulu, S., Nersissian, A., Ohnishi, T., Palmer, G., Que, L., Rousseau, D. L., Sam, J., Scholes, C., Scholes, C. P., Schramm, V. L., Shaka, O., Shanklin, J., Sinclair, P., Singel, D., Stafford, D. W., Stegeman, J., Stubbe, N. J. A., Stubbe, J., Tipton, P. A., Villafranca, N. J. J., Walter, P., Wang, W., Wittaker, J., Zweier, J. L., Belford, L., Beltramini, M., Bowman, M., Chance, M., Clarkson, R., Coffino, A., Crowder, M., Gluck, M. R., Klein, M., Klinman, J., Krzystek, Y., Licht, S., Marcinkeviciene, J., Mcdermott, A., Ming, L., Peisach, J., Ranney, H., Sands, R., Sheik, A., Stubbe, J., Sumuni, R., Tsuneshige, A., Valentine, J., Vieso, V., Wirt, M., Wong, K. & Zhang, C.

    9/30/854/30/00

    Project: Research project