title: Endonuclease and Exonuclease Activities on Oligodeoxynucleotides Containing Spiroiminodihydantoin Depend on the Sequence Context and the Lesion Stereochemistry.
abstract: 8-Oxo-7,8-dihydro-2'-deoxyguanosine (dOG), a well-studied oxidation product of 2'-deoxyguanosine (dG), is prone to facile further oxidation forming spiroiminodihydantoin 2'-deoxyribonucleoside (dSp) in the nucleotide pool and in single-stranded oligodeoxynucleotides (ODNs). Many methods for quantification of damaged lesions in the genome rely on digestion of DNA with exonucleases or endonucleases and dephosphorylation followed by LC-MS analysis of the resulting nucleosides. In this study, enzymatic hydrolysis of dSp-containing ODNs was investigated with snake venom phosphodiesterase (SVPD), spleen phosphodiesterase (SPD) and nuclease P1. SVPD led to formation of a dinucleotide, 5'-d(Np[Sp])-3' (N = any nucleotide) that included the undamaged nucleotide on the 5' side of dSp as the final product. This dinucleotide was a substrate for both SPD and nuclease P1. A kinetic study of the activity of SPD and nuclease P1 showed a sequence dependence on the nucleotide 5' to the lesion with rates in the order dG>dA>dT>dC. In addition, the two diastereomers of dSp underwent digestion at significantly different rates with dSp1>dSp2; nuclease P1 hydrolyzed the 5'-d(Np[Sp1])-3' dinucleotide two- to six-fold faster than the corresponding 5'-d(Np[Sp2])-3', while for SPD the difference was two-fold. These rates are chemically reasoned based on dSp diastereomer differences in thesynvs.antiglycosidic bond orientation. A method for the complete digestion of dSp-containing ODNs is also outlined based on treatment with nuclease P1 and SVPD. These findings have significant impact on the development of methods to detect dSp levels in cellular DNA.
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, U.S.A.
authors.forename: Xin
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, U.S.A.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Fleming
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, U.S.A.
authors.forename: James G
authors.initials: JG
authors.lastname: Muller
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, U.S.A.
authors.forename: Cynthia J
authors.initials: CJ
authors.lastname: Burrows