Project: “Mathematical modeling of repair systems in living organisms”

1. Project: “Mathematical modeling of repair systemsin living organisms” Theoretical investigation of the effect of different initial concentration of8-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 Base excision repair system Escherichia coli bacterial cells 8-oxoguanine (8-oxoG)

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

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

6. Structural model of E. coli BER y1 /Belov, 2010 (in press)/ 8-oxoG e1 υ1 Fpg (GA) y2 AP site e1 e1 Fpg (EA) Fpg (LA) υ2 υ3 y4 y3 3'-nicked site 5'-nicked site υ4 e1 Fpg (PA) GA – glycosylase activity y5 ssDNA EA – endonuclease activity υ5 e2 PolI LA – lyase activity filled gap with two nicks y6 PA – phosphodiesterase activity e3 υ6 AP – apurinic/apyrimidinic site DNA ligase ssDNA – a single-stranded DNA repaired DNA adduct y7 Pol I – DNA polymerase I

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

8. Kinetic parameters estimation

9. Kinetic parameters estimation

10. Concentration-dependent kinetic parameters

11. Modeling biochemical reactions (Gillespie, 1977)

12. y1 8-oxoG e1 υ1 Fpg (GA) y2 AP site e1 Fpg (EA) e1 Fpg (LA) υ2 υ3 y4 y3 3'-nicked site 5'-nicked site υ4 e1 Fpg (PA) y5 ssDNA υ5 e2 PolI filled gap with two nicks y6 e3 υ6 DNA ligase y7 repaired DNA adduct Time, s

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

14. RESULTS for [AP site • Fpg] N, nmol/L N, nmol/L Time, s Time, s 1 µmol/L 2 µmol/L 4 µmol/L

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

16. 1 µmol/L 2 µmol/L RESULTS for [ssDNA • Pol I] N, nmol/L N, nmol/L Time, s Time, s Tim,s 4 µmol/L N, nmol/L Time, s

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

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

19. RESULTS for [Pol I] 1 µmol/L 2 µmol/L N, nmol/L N, nmol/L 0 Time, s Time, s 0 0 Tim,s N, nmol/L 4 µmol/L Time, s

20. Conclusion • The kinetics of base excision repair is modeled for different DNA lesion levels. • For the first time the kinetics of basic intermediate DNA states and BER enzymes are investigated under three different initial concentration of 8-oxoguanine. • For different initial concentrations of 8-oxoguanine, we obtained time shift in the kinetics of all intermediate DNA states and BER enzymes. • 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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