Kinetics and mechanism of the general-acid-catalyzed ring-closure of the malondialdehyde-DNA adduct, N2-(3-oxo-1-propenyl)deoxyguanosine (N2OPdG-), to 3-(2'-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin- 10(3H)-one (M1dG).
3-(2'-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG) is the major product of the reaction of deoxyguanosine with malondialdehyde (MDA). M1dG blocks replication by DNA polymerases in vitro and is mutagenic in vivo. M1dG reacts with hydroxide to form the N2-(3-oxo-1-propenyl)deoxyguanosine anion (N2OPdG-). This reaction is pH-dependent and reverses under neutral and acidic conditions to form M1dG. Here we describe the kinetics and mechanism of the ring-closure reaction in both the nucleoside and oligonucleotides. Kinetic analysis of absorbance and fluorescence changes demonstrates that ring-closure is biphasic, leading to the rapid formation of an intermediate that slowly converts to M1dG in a general-acid-catalyzed reaction. The dependence of the rate of the rapid phase on pH reveals the pKa for protonated N2OPdG is 6.9. One-dimensional 1H NMR and DQF-COSY experiments identified two distinct intermediates, N2OPdG-H and 8-hydroxy-6,7-propenodeoxyguanosine (HO-Prene-dG), that are formed upon acidification of N2OPdG-. Characterization of ring-closure in single-stranded and in melted duplex oligonucleotides shows M1dG formation is also acid-catalyzed in single-stranded oligonucleotides and that the denaturation of an oligonucleotide duplex enhances ring-closure. This work details the complexity of ring-closure in the nucleoside and oligonucleotides and provides new insight into the role of duplex DNA in catalyzing ring-opening and ring-closing of M1dG and N2OPdG.
Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
James N
JN
Riggins
Derek A
DA
Pratt
Markus
M
Voehler
J Scott
JS
Daniels
Lawrence J
LJ
Marnett
D002384:Catalysis; D018736:DNA Adducts / Q000737:chemistry*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011687:Purines / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*; D013816:Thermodynamics
C090855:1,N(2)-(1,3-propano)-2'-deoxyguanosine; D018736:DNA Adducts; D011687:Purines; D011743:Pyrimidines; C107643:pyrimido(1,2-a)purin-10(3H)-one; C089188:3-(2'-deoxyribofuranosyl)pyrimido(1,2-a)purin-10(3H)-one; D003849:Deoxyguanosine