In vitro nucleotide misinsertion opposite the oxidized guanosine lesions spiroiminodihydantoin and guanidinohydantoin and DNA synthesis past the lesions using Escherichia coli DNA polymerase I (Klenow fragment)

Abstract

The low redox potential of 8-oxo-7,8-dihydroguanine (OG), a molecule regarded as a marker of oxidative damage in cells, makes it an easy target for further oxidation. Using a temperature-dependent method of synthesis, the oxidation products of OG, guanidinohydantoin (Gh) and/or its isomer iminoallantoin (Ia) as well as spiroiminodihydantoin (Sp), have been site-specifically incorporated into DNA oligomers. Single nucleotide insertion and primer extension experiments using Escherichia coli Kf exo(-) DNA polymerase were carried out under "standing start" and "running start" conditions in various sequence contexts. dAMP and dGMP were found to be inserted opposite these OG oxidation products. Steady-state kinetic studies show that the Gh/Ia.G base pair yields a lower K(m) value compared to the Sp.G pair or X.A (X = Gh/Ia or Sp). Running start experiments using oxidized and unoxidized OG-containing templates showed enhanced full extension in the presence of all four dNTPs. A sequence preference for efficiency of extension was found when Gh/Ia and Sp are present in the DNA template, possibly leading to primer misalignment. Full extension is more efficient for the templates containing two Gs immediately 3' to the lesions compared to two As. Although these lesions cause a significant block for DNA elongation, results show that they are more easily bypassed by the polymerase when situated in the appropriate sequence context. UV melting studies carried out on duplexes mimicking the template/primer systems were used to characterize thermal stability of the duplexes. These experiments suggest that both Gh/Ia and Sp destabilize the duplex to a much greater extent than OG, with Sp being most severe.