1. Project: “Mathematical modeling of repair systems in living organisms” Theoretical investigation of the effect of different initial concentration of 8-oxoguanine on the base excision repair kinetics Nyathi F. 1, Magonono F.A. 1, Someketa M.A. 2 1 University of Venda,Thohoyandou, South Africa 2 University of Fort Hare, Alice, South Africa Supervisor: Dr. Oleg Belov Assistant: Svetlana Aksenova LRB, JINR

2. Mathematical modeling of repair systems is a key approach to investigate details of the induced mutation process

3. The objects of our research Escherichia coli bacterial cells 8-oxoguanine (8-oxoG) Base excision repair system

4. (γ-radiation, 60 Co ) (Dizdaroglu et al., 1993) (γ-radiation, 60 Co, 55 Gy) 8-oxoguanine is a most common and stable product of oxidative DNA damage under influence of ionizing radiation

5. Fpg-dependent base excision repair /Sugahara et al., 2000/ Formamidopyrimidine-DNA-glycosilase (Fpg protein, MutM protein) Base excision repair

6. y1y1 y2y2 υ1υ1 e1e1 y3y3 y4y4 y5y5 y6y6 y7y7 e3e3 e2e2 υ2υ2 υ3υ3 υ4υ4 υ6υ6 υ5υ5 8-oxoG AP site 5'-nicked site 3'-nicked site ssDNA filled gap with two nicks DNA ligase Fpg (GA) Pol I Fpg (EA) Fpg (LA) Fpg (PA) e1e1 e1e1 e1e1 repaired DNA adduct GA – glycosylase activity EA – endonuclease activity LA – lyase activity PA – phosphodiesterase activity AP – apurinic/apyrimidinic site ssDNA – a single-stranded DNA Pol I – DNA polymerase I Structural model of E. coli BER /Belov, 2010 (in press)/

7. . Stochiometric model of Fpg dependent base excision repair in Escherichia coli bacterial cells /Belov, 2010 (in press)/

8. Kinetic parameters estimation

10. Concentration-dependent kinetic parameters Concentration of 8-oxoG 1 µmol/L2 µmol/L4 µmol/L 9.0 s -1 9.8 s -1 10.0 s -1 0.0641 s -1 0.0781 s -1 0.0472 s -1

11. Modeling biochemical reactions (Gillespie, 1977)

12. y1y1 y2y2 υ1υ1 e1e1 y3y3 y4y4 y5y5 y6y6 y7y7 e3e3 e2e2 υ2υ2 υ3υ3 υ4υ4 υ6υ6 υ5υ5 8-oxoG AP site 5'-nicked site 3'-nicked site ssDNA filled gap with two nicks DNA ligase Fpg (GA) Pol I Fpg ( EA ) Fpg (LA) Fpg (PA) e1e1 e1e1 e1e1 repaired DNA adduct Time, s

13. N, nmol/L Time, s N, nmol/L [8-oxoG] [8-oxoG Fpg] 1µmol/L 2 µmol/L 4 µmol/L

14. N, nmol/L Time, s N, nmol/L 1 µmol/L 2 µmol/L 4 µmol/L Time, s

15. N, nmol/L Time, s N, nmol/L [3′-nicked site Fpg ] [5′-nicked site Fpg] 1 µmol/L 2 µmol/L 4 µmol/L

16. N, nmol/L Time, s Tim,s Time, s N, nmol/L Time, s N, nmol/L 1 µmol/L 2 µmol/L 4 µmol/L

17. Time, s N, nmol/L [filled gapDNA ligase] 1 µmol/L 2 µmol/L 4 µmol/L Time, s [ repaired DNA adduct] N, nmol/L

18. Time, s N, nmol/L [Fpg] [DNA ligase] 1µmol/L 2 µmol/L 4 µmol/L

19. Time, s Tim,s Time, s N, nmol/L 0 0 Time, s 1 µmol/L 2 µmol/L 4 µmol/L 0 N, nmol/L

20. Conclusion 1.The kinetics of base excision repair is modeled for different DNA lesion levels. 2.For the first time the kinetics of basic intermediate DNA states and BER enzymes are investigated under three different initial concentration of 8-oxoguanine. 3.For different initial concentrations of 8-oxoguanine, we obtained time shift in the kinetics of all intermediate DNA states and BER enzymes. 4.On the basis of the obtained results, it can be concluded that Fpg protein and DNA ligase demonstrate multi- turnover kinetics during BER.

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