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Book of Abstracts: Albany 2011

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

Characterization of Fpg Protein Binding to DNA Lesions Using Pyrrolocytosine Fluorescence

Reactive oxygen species damage DNA to produce a variety of genotoxic lesions. In particular, 7,8-dihydro-8-oxoguanine (oxoG) is one of the most common pre-mutagenic products of base oxidation in DNA. OxoG is repaired (1) through excision by formamidopyrimidine-DNA glycosylase (Fpg) in bacteria or 8-oxoguanine-DNA glycosylase (OGG1) in eukaryotes. In addition to its glycosylase activity, Fpg possesses an AP-lyase activity, which catalyzes sequential elimination of the 3’-phosphate (β-elimination) and the 5’-phosphate (δ-elimination) at the nascent or pre-formed abasic (AP) site, producing a one-nucleotide gap flanked by two phosphates (2). The glycosidic bond breakage is initiated by a nucleophilic attack at C1’ by the Pro-1 residue, resulting in a covalent enzyme–DNA Schiff base intermediate, which then rearranges and undergoes elimination. The three-dimensional structure of E. coli Fpg shows that DNA binding is accompanied with drastic conformational changes, including DNA bending, eversion of oxoG from DNA, and insertion of Met-73, Arg-108 and Phe-110 residues into DNA (3, 4).

In our previous studies we have used quench-flow technique to show that the kinetics of processing of oxoG and AP site lesions by Fpg from E. coli involves a burst and a stationary phases. The data from stopped-flow kinetics with tryptophan and 2-aminopurine fluorescence detection revealed that both the protein and the damaged DNA undergo extensive conformational changes in the course of DNA substrate binding and cleavage (5, 6). It was concluded that the cleaved product formation is initially reversible. We have also applied mass spectrometry with electrospray ionization to follow appearance and disappearance of transient covalent intermediates between Fpg and the substrate DNA (6, 7). The overall rate-limiting step of the enzymatic reaction seemed to be the release of Fpg from its adduct with the 4-oxo-2-pentenal remnant of the deoxyribose moiety formed as a result of DNA strand cleavage by β,δ-elmination.

To gain a deeper insight into mechanism by which Fpg protein recognizes DNA lesions we have studied the changes both in fluorescence of pyrrolocytosine (pyrC) (Fig. 1A) and in FRET of Trp/pyrC donor/acceptor pair. PyrC was placed opposite damaged nucleotides in DNA strand.



Fig. 1. Structure of pyrC (A) and its fluorescence traces in the Fpg catalytic cycles with oxoG-, AP- and tetrahydrofurane (F) containing DNA-substrates.

Stopped-flow fluorescence kinetics demonstrated the multi-step character of lesion recognition (Fig. 1B). Thermodynamic parameters of each recognition step were found by analysis of fluorescence traces at different temperatures.

Acknowledgements

This work was supported by grants from the RFBR (10-04-00070), Siberian Division of the Russian Academy of Sciences (28, 48), Russian Ministry of Education and Sciences (02.740.11.0079, NS-3185.2010.04, MK-1304.2010.04).

References

  1. 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).
  2. D. O. Zharkov, G. Shoham, A. P. Grollman, A. P. DNA Repair 2, 839-862 (2003).
  3. R. Gilboa, D. O. Zharkov, G. Golan, A. S. Fernandes, S. E. Gerchman, E. Matz, J. H. Kycia, A. P. Grollman, G. Shoham, J. Biol. Chem. 277, 19811-19816 (2002).
  4. 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).
  5. N. A. Kuznetsov, V. V. Koval, D. O. Zharkov, Y. N. Vorobjev, G. A. Nevinsky, K. T. Douglas, K, O. S. Fedorova, Biochemistry 46, 424-435 (2007).
  6. N. A. Kuznetsov, D. O. Zharkov, V. V. Koval, M. Buckle, O. S. Fedorova, Biochemistry 48, 11335-11343 (2009).
  7. V. V. Koval, N. A. Kuznetsov, A. A. Ishchenko, M. K. Saparbaev, O. S. Fedorova, Mutation Res. 685, 3-10 (2010).

Nikita A. Kuznetsov
Olga S. Fedorova*

Institute of Chemical Biology and Fundamental Medicine
Novosibirsk 630090, Russia

ph: +7-383-363-5175
fx: +7-383-363-5153
fedorova@niboch.nsc.ru