Project: “Mathematical modeling of repair systems
in 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 Africa2 University of Fort Hare, Alice, South Africa
Supervisor: Dr. Oleg BelovAssistant: Svetlana AksenovaLRB, JINR
Mathematical modeling of repair systems is a key approach to investigate details of the induced mutation process
The objects of our research
Escherichia colibacterial cells
8-oxoguanine(8-oxoG)
Base excision repair system
(γ-radiation, 60Co )
(Dizdaroglu et al., 1993)
(γ-radiation, 60Co, 55 Gy)
8-oxoguanine is a most common and stable product of oxidative DNA
damage under influence of ionizing radiation
Fpg-dependent base excision repair
/Sugahara et al., 2000/
Formamidopyrimidine-DNA-glycosilase
(Fpg protein, MutM protein)
Base excision repair
y1
y2
υ1 e1
y3y4
y5
y6
y7
e3
e2
υ2 υ3
υ4
υ6
υ5
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)
e1 e1
e1
repaired DNA adduct
GA – glycosylase activityEA – 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)/
.
Stochiometric model of Fpg dependent base excision repair in Escherichia coli
bacterial cells
/Belov, 2010 (in press)/
Kinetic parameters estimation
Kinetic parameters estimation
Concentration-dependent kinetic parameters
Concentration of 8-oxoG 1 µmol/L 2 µmol/L 4 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
Modeling biochemical reactions
(Gillespie, 1977)
y1
y2
υ1 e1
y3y4
y5
y6
y7
e3
e2
υ2 υ3
υ4
υ6
υ5
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)
e1 e1
e1
repaired DNA adduct
Time, s
N, n
mol
/LTime, sTime,
sTime, s
N, n
mol
/L
[8-oxoG] [8-oxoG • Fpg]
1µmol/L 2 µmol/L
4 µmol/L
N, n
mol
/LTime, s
N, n
mol
/L
1 µmol/L2 µmol/L
4 µmol/L
Time, s
N, n
mol
/LTime, sTime, s
N, n
mol
/L
[3′-nicked site
•Fpg]
[5′-nicked site •Fpg]
1 µmol/L2 µmol/L
4 µmol/L
N, n
mol
/L
Time, s
Tim,s
Time, s
N, n
mol
/L
Time, s
N, n
mol
/L
1 µmol/L 2 µmol/L
4 µmol/L
Time, s
N, n
mol
/L[filled gap•DNA ligase]
1 µmol/L
2 µmol/L
4 µmol/L
Time, s
[repaired DNA adduct]
N, n
mol
/L
N, n
mol
/L
N, n
mol
/L
Time, sTime, s
N, n
mol
/L
[Fpg] [DNA ligase]
1µmol/L2 µmol/L
4 µmol/L
Time, s
Tim,s
Time, s
N, n
mol
/L
N, n
mol
/L
0 0
Time, s
1 µmol/L 2 µmol/L
4 µmol/L
0
N, n
mol
/L
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.
THANK YOUFOR YOUR ATTENTION!
СПАСИБО ЗА ВНИМАНИЕ!