“Mirror” Method to Estimate Mutagenic Activity of DNA Lesions

Abstract

Abstract:We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5 -monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains ("mirror" products) using a template containing 8-oxoG. The misincorporation of dAMP in the "mirror" product forms EcoRI sites. The restriction analysis of double-stranded DNAs obtained by PCR of "mirror" product allows quantification of the mutagenesis frequency. In addition, single-strand conformational polymorphism (SSCP) analysis of the single-stranded "mirror" products shows that different DNA polymerases only incorporate dA or dC opposite 8-oxoG. The proposed approach used in developing this technique can be applied in the study of other lesions as well, both single and clustered.

Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method

“…Earlier, we proposed a "mirror' method for studying the mutagenic activity of purified DNA polymerases [1]. In the present work, to estimate the mutagenic activity of 8-oxoG in nuclear extracts, we modified a "mirror" method and called it "framed mirror" method.…”

“…One of the main causes of GC-AT transversion somatic mutations is a DNA lesion, 8-oxoguanine (8-oxoG) arising in the genome under the action of reactive oxygen species (ROS) generated in cells in the processes of oxidative phosphorylation [1,2]. The intensity of the conversion of this DNA lesion into a mutation is a key moment of aging and oncological, neurological, and some other diseases [3,4].…”

“…Recently we proposed a "mirror" method to study the mutagenic potential of 8-oxoG using purified DNA polymerases [1]. At the first stage of this method, similar to the method used by Targgart et al [12], a specific DNA polymerase is used to obtain a copy of the template that we called "mirror".…”