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.

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