RESUMO
Modified DNA bases are widespread in nature. They can be found in eukaryotes, prokaryotes, and bacteriophages. They may completely replace the standard base or replace only a small fraction. Their substituents vary from simple methyl or hydroxy groups to large moieties like amino acids and multiply hexosylated side chains. This review gives an overview of the modified DNA bases identified thus far, with emphasis on the "very unusual" or hypermodified DNA bases. Although these have been detected mainly in bacteriophages, recent work has shown the presence of a novel hypermodified DNA base in the eukaryote Trypanosoma brucei. We speculate on the biosynthesis and function of this novel base beta-D-glucosyl-hydroxymethyluracil.
Assuntos
DNA/química , Glucosídeos/química , Purinas/química , Pirimidinas/química , Uracila/análogos & derivados , Animais , Bacteriófagos/química , Bacteriófagos/metabolismo , DNA/biossíntese , DNA Viral/biossíntese , DNA Viral/química , Células Eucarióticas/química , Células Procarióticas/química , Purinas/biossíntese , Pirimidinas/biossíntese , Trypanosoma brucei brucei/química , Uracila/químicaRESUMO
We have previously shown that the DNA of the unicellular eukaryote T. brucei contains about 0.1% of a novel modified base, called J. The presence of J correlates with a DNA modification associated with the silencing of telomeric expression sites for the variant surface antigens of trypanosomes. Here we show that J is 5-((beta-D-glucopyranosyloxy)-methyl)-uracil (shortened to beta-D-glucosyl-hydroxymethyluracil), a base not previously found in DNA. We discuss putative pathways for the introduction of this base modification at specific positions in the DNA and the possible contribution of this modification to repression of surface antigen gene expression.
Assuntos
DNA de Protozoário/química , Glucosídeos/análise , Trypanosoma brucei brucei/química , Uracila/análogos & derivados , Animais , Cromatografia Líquida , Cromatografia em Camada Fina , DNA de Protozoário/isolamento & purificação , Desoxirribonucleotídeos/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Galactosiltransferases , Cromatografia Gasosa-Espectrometria de Massas , Expressão Gênica , Glucosídeos/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Telômero/fisiologia , Trypanosoma brucei brucei/genética , Uracila/análise , Uracila/química , Glicoproteínas Variantes de Superfície de Trypanosoma/biossínteseRESUMO
A method for sensitive analysis of the oxidatively modified nucleosides 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 5-hydroxymethyl-2'-deoxyuridine (HMdU) is described. The method combines acetylation and pentafluorobenzylation of the nucleosides followed by analysis by gas chromatography/electron capture negative ion chemical ionization-mass spectrometry. The detection limits of the method for aqueous standards of HMdU and 8-OHdG were 12 and 18 fmol of starting material (signal-to-noise ratio, 3:1), respectively. The method was linear for 8-hydroxy-2'-deoxyguanosine over five orders of magnitude and gave satisfactory reproducibilities (intraassay RSD < or = 5%) for the analysis of 8-OHdG in both aqueous standards and urine fortified at the level of 35 nM. The limit of detection for the analysis of 8-OHdG in urine was 1.8 pmol, corresponding to a level of 8-OHdG in urine of 35 nM (10 micrograms/liter) at a urine sample volume of 50 microliters. Besides urine the method was applied to the analysis of 5-hydroxy-methyl-2'-deoxyuridine isolated from genomic DNA of Bacillus subtilis bacteriophage H1. Results obtained indicate that the method is potentially suitable for the determination of oxidized nucleosides in biological samples. The selectivity of the method should be enhanced in order to lower the limit of detection in biological samples.
Assuntos
Desoxiguanosina/análogos & derivados , Timidina/análogos & derivados , 8-Hidroxi-2'-Desoxiguanosina , Acetilação , Bacillus subtilis , Bacteriófagos/genética , Bioensaio , Catálise , DNA Viral/isolamento & purificação , Desoxiguanosina/análise , Desoxiguanosina/urina , Fluorbenzenos , Cromatografia Gasosa-Espectrometria de Massas , Modelos Lineares , Oxirredução , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Timidina/análiseRESUMO
We have previously reported the detection of two unusual nucleotides, pdJ and pdV, in the DNA of Trypanosoma brucei (Gommers-Ampt et al., 1991). pdJ was found to be a novel nucleotide and is possibly involved in the regulation of variant specific surface antigen gene expression in trypanosomes. Recent evidence suggests that V could be a precursor of J, making V a key compound in the study of the biosynthesis and function of J. We have therefore determined the structure of V and here we present proof that V is HOMeU. The identity is based on a detailed comparison of dV(p) with authentic HOMedU(p), showing: I) co-migration in three different liquid chromatography analyses II) identical UV absorbance characteristics III) identical behavior in acetyl-pentafluorobenzyl derivatization and subsequent Gas chromatography/Mass spectrometry (GC/MS). The GC/MS technique has not been used before to analyse HOMedU purified from biological material. Because of its high sensitivity, it may also be useful for the detection of the low amounts of HOMedU resulting from oxidative damage of DNA.
