Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

Conformational Dynamics of Interactions of Escherichia coli Endonuclease VIII with DNA Substrates

Free radical-mediated damage is believed to be the most widespread type of DNA damage and includes modifications to purine and pyrimidine bases and deoxyribose moieties. Base excision repair (BER) is the major pathway that processes oxidative DNA lesions. BER is initiated by DNA glycosylases that recognize and remove damaged bases from DNA.

DNA repair enzymes are faced with the difficult task of recognizing their substrates in a large excess of unmodified DNA. Two questions are frequently asked about these enzymes: how do they find the damaged sites in DNA and how do they discriminate between damaged and undamaged sites. Structural data suggest that conformations of DNA repair enzymes and their substrate DNA are often changed in the ES complex in comparison with the free enzyme and substrate molecules. Therefore, it can be reasonably suggested that these conformational changes are responsible for the specificity of BER enzymes.

Previously, we reported the study of the dynamics of conformational transitions of repair processes catalyzed by two unrelated 8-oxoguanine DNA glycosylases, E. coli Fpg protein and human OGG1 (1-5). Here, we apply the same type of pre-steady-state conformational analysis to address the conformational changes accompanying DNA binding and damage recognition by E. coli endonuclease VIII (Nei). Nei is a bacterial DNA glycosylase/AP lyase that excises oxidatively modified pyrimidines from DNA. During catalysis, the N-terminal proline moiety of Nei attacks the C1? atom of the damaged nucleoside, leading to the loss of the base and the formation of a Schiff-base intermediate. The latter rearranges to eliminate the 3?-phosphate (β-elimination) and 5?-phosphate (δ-elimination) from the deoxyribose moiety.

In our work, a stopped-flow approach combined with the intrinsic tryptophane fluorescence detection have been used to study conformational transitions in Nei. We have used fluorescence kinetics to directly analyze the individual steps of enzyme-DNA complex formation, measure the rate constants along the reaction pathway, and detect kinetic intermediates. Duplex oligonucleotides containing 5,6-dihydrouracil (dHU), natural abasic site, or tetrahydrofuran, an uncleavable abasic site analog were used as DNA substrates. Under single-turnover conditions, at least five conformational transitions were detected in the Nei protein during the catalytic cycle (Fig. 1) responsible for recognition and conversion of substrate. Rate constants for the individual reaction steps were obtained by global fitting procedure.

Figure 1: Changes of Trp fluorescence intensity during interaction of OGG1 with dHU-substrate.

The structure of free Nei and Nei in the complex with DNA (6, 7) can be used to rationalize the observed conformational transitions. Upon DNA binding, the enzyme?s conformation changes from the open form to a more compact closed form, which is likely reflected in the first equilibrium step of the reaction scheme, E + S ⇔ ES1. The damaged base is recognized in a pocket in the Nei globule after being everted from the DNA duplex, and the void created in the duplex by this eversion is filled with the enzyme?s loop Gln69-Tyr71. The eversion and insertion processes are separated in time in most DNA glycosylases and, thus, may be observed as distinct steps corresponding to the equilibria ES1 ⇔ ES2 and ES2 ⇔ ES3. Alternatively, one of these equilibria could be invisible to tryptophan fluorescence, and the ES2 ⇔ ES3 step may correspond to the isomerization of the active site pocket before base excision. As in Fpg, all chemical steps of catalysis (base excision, β- and δ-elimination) coalesce into a single fluorescently discernible step ES3 → EP and, thus, only one of them is likely to be accompanied with significant conformational transitions. The reaction scheme established in this work falls in line with those known for other DNA glycosylases (8) and can serve as a reference point to dissect the origins of Nei specificity for different substrates.


This research was made possible in part by grants from the Wellcome Trust (UK) (070244/Z/03/Z), the Presidium of the Russian Academy of Sciences (MCB Program, 10.5 and 10.6), RFBR (05-04-48619, 07-04-00191), CRDF (Y1-B-08-16, Y2-B-08-08), and INTAS (05-109-4159).

References and Footnotes
  1. Fedorova, O. S., Nevinsky, G. A., Koval, V. V., Ishchenko, A. A., Vasilenko, N. L., Douglas, K. T. Biochemistry 41, 1520-1528 (2002).
  2. Koval, V. V., Kuznetsov, N. A., Zharkov, D. O., Ishchenko, A. A., Douglas, K. T., Nevinsky, G. A., Fedorova, O. S. Nucleic Acids Res 32, 926-935 (2004).
  3. Kuznetsov, N. A., Koval, V. V., Zharkov, D. O., Nevinsky, G. A., Douglas, K. T., Fedorova, O. S. Nucleic Acids Res 33, 3919-3931 (2005).
  4. Kuznetsov, N. A., Koval, Nevinsky, G. A., Douglas, K. T., Zharkov, D. O., Fedorova, O. S. J Biol Chem 282, 1029-1038 (2007).
  5. Kuznetsov, N. A., Koval, V. V., Zharkov, D. O., Vorobiev, Y. N., Nevinsky, G. A., Douglas, K. T., Fedorova, O. S. Biochemistry 47, 424-435 (2007).
  6. Zharkov, D. O., Golan, G., Gilboa, R., Fernandes, A. S., Gerchman, S. E., Kycia, J. H., Rieger, R. A., Grollman, A. P., Shoham, G. EMBO J 21, 789-800 (2002).
  7. Golan, G., Zharkov, D. O., Feinberg, H., Fernandes, A. S., Zaika, E. I., Kycia, J. H., Grollman, A. P., Shoham, G. Nucleic Acids Res 33, 5006-5016 (2005).
  8. Zharkov, D. O., Grollman, A. P. Mutat Res 577, 24-54 (2005).

N. A. Kuznetsov
D. O. Zharkov
O. S. Fedorova*

Institute of Chemical Biology and Fundamental Medicine
Novosibirsk State University
Novosibirsk 630090, Russia

*Email: fedorova@niboch.nsc.ru