Book of Abstracts: Albany 2005

category image Volume 22
No. 6
June 2005

Multiple Conformational Changes in DNA Repair Enzymes During Substrate Recognition and Catalysis

Multiple conformational changes are a general feature of enzymatic mechanisms. Enzyme structure can be pre-formed for substrate recognition or can be optimized for each step in the reaction sequence. In the latter case the enzyme plays a dynamic role in the catalytic process by directly coupling conformational changes throughout the macromolecule to the catalytic process. Clearly the conformational adaptability of the macromolecule is essential for enzyme functions.

We have studied the dynamics of conformational transitions of repair processes catalyzed by the 8-oxoguanine DNA glycosylases, E. coli Fpg-protein and human hOgg1. Working against damaged DNA, these enzymes remove 8-oxoguanine (oxoG) and some other oxidatively damaged bases, formed by reactive oxygen species (ROS). ROS are continuously generated as by-products of respiration, inflammatory and pathological processes or through reaction pathways initiated by UV or ionizing irradiation. Of the oxidatively damaged bases, which are pre-mutagenic and carcinogenic, oxoG is considered as the main lesion. Fpg-protein and hOgg1 are bifunctional DNA N-glycosylases/AP-lyases. DNA glycosylase activity results in hydrolysis of the N-glycosidic bond and formation of an AP-site. AP-lyase activity leads to nicking the phosphodiester backbone of DNA at the site of the lesion through β-elimination. Base excision by Fpg is followed by δ-elimination steps, resulting in a single nucleotide gap flanked by phosphate termin(i).

Thermodynamic analysis of Fpg and hOgg1?s interactions with DNA substrates was carried out using the method of stepwise increase of substrate complexity (SILC) (1) to estimate relative contributions of individual nucleotide units of lesion-containing DNA to its total affinity for these enzymes. Formation of the primary complex between the enzymes and DNA was analyzed by SILC according to the scheme: ortho-phosphate → mononucleotide → single-stranded (ss) non-specific homo-ODN → ss specific hetero-ODN → ds non-specific homo-ODN → ds specific hetero-ODN.

The relative affinities of enzymes for specific and nonspecific oligodeoxynucleotides differ by no more than 2-3 orders of magnitude. Complex formation of enzymes with DNA containing 8-oxoG cannot alone provide the major part of the enzyme specificity, which lies in the kcat term; the reaction rate being increased by 6-8 orders of magnitude by the transition from nonspecific to specific oligodeoxynucleotides (2, 3).

Pre-steady-state kinetics of 8-oxoguanine DNA glycosylase reacting with duplex oligonucleotide substrates containing 8-oxoguanosine, natural abasic site, or tetrahydrofuran, an uncleavable abasic site analog were studied by stopped-flow fluorescence. Under single-turnover conditions multiple transient changes in enzyme fluorescence were observed, indicating sequential conformational transitions in the both proteins (4-6). In the case of hOgg1, the interaction with a substrate containing an aldehydic AP site was very slow, but addition of 8-bromoguanine greatly accelerated the reaction. These data confirmed the published role (7) of the excised 8-oxoguanine in product-assisted catalysis by hOgg1. Rate constants for binding and catalytic stages, determined by global fitting, provide evidence that several fast subsequent conformational changes occur in both enzymes after binding to the substrate, transforming the protein into a catalytically active state. All these results demonstrate the coupling of conformational changes in both 8-oxoguanine DNA glycosylases with substrate recognition and processing.


Supported by grants from the Wellcome Trust (UK) (070244/Z/03/Z), Molecular and Cell Biology Program (10.5), RFBR (04-04-48171, 05-04-48619), the Russian Ministry of Education and Science (NS-1419.2003.4, PD02-1.4-469), CRDF (REC-008, Y1-B-08-16). V. K. was supported by Post Doctoral Fellowship from INTAS (04-83-3849).

References and Footnotes
  1. G. A. Nevinsky. In: Protein Structures. Kaleidoscope of Structural Properties and Functions, pp. 133-222. Ed., V. N. Uversky. Research Signpost (2003).
  2. A. A. Ishchenko, N. L. Vasilenko, O. I. Sinitsina, V. I. Yamkovoy, O. S. Fedorova, K. T. Douglas, and G. A. Nevinsky. Biochemistry 41, 7540-7548 (2002).
  3. A. A. Ishchenko, V. V. Koval, O. S. Fedorova, K. T. Douglas, and G. A. Nevinsky. J. Biomol. Struct. Dyn. 17, 301-310 (1999).
  4. O. S. Fedorova, G. A. Nevinsky, V. V. Koval, A. A. Ishchenko, N. L. Vasilenko, and K. T. Douglas. Biochemistry 41, 1520-1528 (2002).
  5. V. V. Koval, N. A. Kuznetzov, D. O. Zharkov, A. A. Ishchenko, K. T. Douglas, G. A. Nevinsky, and O. S. Fedorova. Nucleic Acids Res. 32, 926-935 (2004).
  6. N. A. Kuznetsov, V. V. Koval, D. O. Zharkov, G. A. Nevinsky, K. T. Douglas, and O. S. Fedorova. Nucleic Acids Res. (2005) submitted.
  7. J. C. Fromme, S. D. Bruner, W. Yang, M. Karplus, and G. L. Verdine. Nat. Struct. Biol. 10, 204-211 (2003).

O. Fedorova1,*
G. Nevinsky1
V. Koval1
D. Zharkov1
N. Kuznetsov1
K. T. Douglas2

1Institute of Chemical Biology & Fundamental Medicine
Novosibirsk State University
Novosibirsk 630090, Russia
2School of Pharmacy and Pharmaceutical Sciences
University of Manchester
Manchester, M13 9PL, UK

*Phone: +7 (383) 3309274
Fax: +7 (383) 3333677
Email: fedorova@niboch.nsc.ru