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Book of Abstracts: Albany 2011

category image Albany 2011
Conversation 17
June 14-18 2011
©Adenine Press (2010)

Kinetic Characterization of the Repair Enzyme OGG1 acting on Triplet Repeat DNA

Triplet repeat sequences, such as CAG/CTG, expand in the human genome to cause several neurological disorders. Work from other laboratories has implicated the DNA repair enzyme 8-oxo-7,8-dihydroguanine glycosylase (OGG1) in triplet repeat expansion (1, 2). OGG1 initiates the base excision repair (BER) process in mammalian cells by excising the oxidatively damaged nucleobase 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Then AP (Apurinic/apyrimidinic) endonuclease (3) nicks DNA hydrolytically 5’ to the AP site so that the repair can be completed in the subsequent steps by DNA polymerase and DNA ligase. Motivated by the demonstrated involvement of OGG1 in triplet repeat expansion we initiated a comprehensive kinetic analysis of the activity of OGG1 on triplet repeat oligonucleotide substrates. These substrates include canonical CAG/CTG duplexes and also the non-canonical stem-loop hairpins that are proposed to contribute to repeat expansion. We determined the binding affinity, the rate of cleavage of the N-glycosidic bond of 8-oxoG, and the rate of product release for human OGG1 acting on TNR sequences. Comparison of the results obtained for the triplet repeats sequences to those obtained for a mixed-sequence duplex allowed us to define the contribution of sequence context of the damage to enzyme activity. We demonstrate that the structure of the DNA substrate modulates OGG1 activity whereas changes to the sequence composition are well tolerated. Taken together, these results contribute to our knowledge of the sequence and structural specificity of OGG1 and, furthermore, define the kinetic parameters of the event that initiates triplet repeat expansion via BER.

References

  1. I. V. Kovtun, Y. Liu, M. Bjoras, A. Klungland, S. H. Wilson, and C. T. McMurray, Nature 447, 447-452 (2007).
  2. Y. Liu, R. Prasad, W. A. Beard, E. W. Hou, J. K. Horton, C. T. McMurray, and S. H. Wilson, J. Biol. Chem. 284, 28352-28366 (2009).
  3. N. A. Timofeyeva, V. V. Koval, D. G. Knorre, D. O. Zharkov, M. K. Saparbaev, A. A. Ishchenko, O. S. Fedorova, J Biomol Struct Dyn 26, 637-652 (2009).

Daniel A. Jarem
Kelly M. Schermerhorn
Nicole R. Wilson
Sarah Delaney

Department of Chemistry
Bown University
324 Brook St, Box H
Providence RI 02912

ph: 401-863-3590
fx: 401-863-9368
sarah_delaney@brown.edu