No abstract

[reaction: see text] Further oxidation of the common DNA lesion 8-oxo-7,8-dihydroguanosine by one-electron oxidants such as IrCl6(2-), Fe(CN)6(3-), or SO4-* leads to two major products, depending upon reaction conditions. In nucleosides at pH 7, 22 degrees C, the principal product is shown herein to be a spiroiminodihydantoin nucleoside, as a diastereomeric mixture, that can be characterized by NMR, ESI-MS/MS, and independent synthesis.

Use of one-electron oxidants such as Na(2)IrCl(6) to oxidize 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) residues in oligodeoxynucleotides was previously shown to lead to predominant formation of a base lesion of mass M - 10 compared to starting material [Duarte et al. (1999) Nucleic Acids Res. 27, 596-502]. To thoroughly characterize the structure of this lesion, the oxidation of the nucleoside 9-N-(2',3',5'-tri-O-acetyl-beta-D-erythro-pentanosyl)-8-oxo-7,8-dihydroguanine with one-electron oxidants at pH 2-4 was used as a model for duplex DNA oxidation of OG residues. (1)H NMR and H,H COSY NMR studies in CD(3)OD along with LC-ESI-MS/MS fragmentation analysis are consistent with the assignment of the M - 10 species as a mixture of two pH-dependent equilibrating isomers, a guanidinohydantoin (Gh) and an iminoallantoin (Ia) nucleoside, both present as mixtures of epimers at the C5 position of the hydantoin ring, i.e., four total isomers are formed. The Gh/Ia mixture is formed from hydration and decarboxylation of the initially formed intermediate 5-hydroxy-8-oxo-7,8-dihydroguanosine, a species that is also produced by four-electron oxidation (e.g., singlet oxygen) of guanosine. The product mixture can be further oxidized to a species designated Ia(ox), a hydrolytically unstable material at pH 7 that has been characterized by ESI-MS and (1)H NMR. Competition studies with 8-oxo-7,8-dihydroadenosine placed the redox potential of Gh/Ia at about 1.0 V vs NHE. These studies provide important information concerning the structures of lesions obtained when OG, a "hot spot" for oxidative damage, serves as a "hole trap" in long-range electron-transfer studies.

Isotope dilution gas chromatography-mass spectrometry analysis of genomic DNAs isolated from the different growth phases of Neurospora crassa revealed significant differences in the amounts of 5-methylcytosine; the mol% of 5-methylcytosine was 0.36 in conidia (asexual spores), 0.40 in conidial germlings cultured for 3 h, 0.24 in mycelial cells that had grown exponentially for 6 and 12 h, and 0.40 in stationary-phase mycelial cells. These results indicate an approximate inverse correlation between the level of C methylation in the DNA and the presumed level of developmentally related transcriptional activity.

Isotope dilution gas chromatography-mass spectrometry analysis of genomic DNAs isolated from three Candida albicans isolates showed significant differences in the amounts of 5-methyldeoxycytidine (msCyt) in DNA from yeast-form and from mnycelial-form cells; the moles percent m5Cyt were 0.11, 0.11, and 0.097 for yeast-form DNA and 0.045, 0.053, and 0.047 for mycelial-form DNA for the three isolates Sh8, 9938, and In eucaryotes, DNA methylation and 5-methyldeoxycytidine (m5Cyt) in particular have been shown to affect many important biological functions through effects on gene expression, and for many eucaryotic organisms a negative correlation between m5Cyt levels and gene expression has been reported (for reviews, see references 1, 4, 5, 8, and 11). In this study we investigated (i) whether C. albicans possesses significant levels of m5Cyt in its genomic DNA and (ii) whether differences in m5Cyt levels could be found in DNA from yeast-and mycelial-form cells. Quantitative analysis of cytosine methylation in DNA was done by the very sensitive and selective stable-isotope dilution gas chromatographymass spectrometry procedure (3).Three C. albicans isolates were used in the study: 9938 was originally obtained as a human isolate and is maintained in our culture collection; Sh8 was supplied by David Stevens, Santa Clara Valley Medical Center, San Jose, Calif., and B311 (7) Fig. 1. Chromatographic base-line fluctuation in the high-sensitivity m5Cyt channel led to some uncertainty in the establishment of peak height.