DNA Polymerase ν Rapidly Bypasses O6-Methyl-dG but Not O6-[4-(3-Pyridyl)-4-oxobutyl-dG and O2-Alkyl-dTs

Abstract

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco carcinogen that forms mutagenic DNA adducts including O⁶-methyl-2'-deoxyguanosine (O⁶-Me-dG), O⁶-[4-(3-pyridyl)-4-oxobut-1-yl]-dG (O⁶-POB-dG), O²-methylthymidine (O²-Me-dT), and O²-POB-dT. We evaluated the ability of human DNA polymerase ν to bypass this damage to evaluate the structural constraints on substrates for pol ν and to evaluate if there is kinetic evidence suggesting the in vivo activity of pol ν on tobacco-induced DNA damage. Presteady-state kinetic analysis has indicated that O⁶-Me-dG is a good substrate for pol ν, while O⁶-POB-dG and the O²-alkyl-dT adducts are poor substrates for pol ν. The reactivity with O⁶-Me-dG is high with a preference for dCTP > dGTP > dATP > dTTP. The catalytic activity of pol ν toward O⁶-Me-dG is high and can potentially be involved in its bypass in vivo. In contrast, pol ν is unlikely to bypass O⁶-POB-dG or the O²-alkyl-dTs in vivo.

Figure 1

Potential base pair structures.

Figure 2

Relative k_cat/K_m values for the single nucleotide incorporation opposite (A) dG(closed circle), O⁶-Me-dG (open square), and O⁶-POB-dG (closed triangle), and (B) dT (closed circle), O²-Me-dT (open square), and O²-POB-dT (closed triangle).

Figure 3

Incorporation of single dNTPs opposite templates containing (A) dG and (dT) and analogues. The concentration of the DNA was 15 nM, the pol 150 nM, and the dNTP was 50 μM.

Figure 4

Analysis of the time course data. (A) Incorporation of pol ν (150 nM) catalyzed dCTP (10, square; 20, triangle; 50, upside down triangle; 100, diamond; and 200, circle μM) incorporation opposite DNA containing O⁶-Me-dG (15 nM). The lines are the best fit to equation 1. The error bars represent the standard deviation of three determinations. (B) Plot of amplitude (A) and burst rate constant (k) versus dCTP concentration. The lines are the best fit to eq 2, and the error bars are the standard errors.

Figure 5

Relative k_pol/K_d values for the single nucleotide incorporation opposite (A) dG(closed circle), O⁶-Me-dG (open square), O⁶-POB-dG (closed triangle), and (B) dT (closed circle), O²-Me-dT (open square), and O²-POB-dT (closed triangle).

Chart 1

Chart 2

Oligodeoxynucleotide sequences