Studies of base modification in deoxyribonucleic acid by mass spectrometry.

Mass spectrometry has been applied to two types of problems relating to the occurrence of modified bases in deoxyribonucleic acid (DNA), in which small sample amounts have dictated the requirement for a highly sensitive analytical method. One problem concerns accurate measurement of levels of occurrence of known modified bases, and the other illustrates determination of the structure of an uncharacterized modified deoxyribonucleoside. A direct chemical assay for 5-methylcytosine in DNA has been developed based on stable isotope dilution gas chromatography - mass spectrometry with selected ion monitoring. 5-Methylcytosine is determined relative to thymine using 5-({('2)H(,3)}methyl)cytosine and {methyl-('2)H(,3)}thymine. The method yields quantitative measurements down to 0.002 mol %, or approximately 1 5-methylcytosine in 50,000 bases, based on measurement of 500 nanograms of DNA. The method has been applied to analysis of DNAs of very low or uncharacterized 5-methylcytosine content. DNAs of Drosophila melanogaster, Aedes albopictus, Sulfolobus solfataricus, Methanococcus voltae, and two strains of yeast, were found not to contain 5-methylcytosine to an extent of greater than 1 in 50,000 bases. Halobacterium volcanii DNA was found to contain 0.24% 5-methylcytosine relative to thymine content. The origin of 5-methylcytosine in bacteriophage (PHI)X-174 DNA was also investigated. The new assay was used to analyze both single-stranded (virion) and double-stranded RF I forms of (PHI)X-174 DNA isolated from phage propagated in E. coli C lacking the DNA cytosine methylase (dcm('-)), and in wild-type E. coli C (dcm('+)). Both forms of phage DNA from E. coli C (dcm('-)) were found to contain 5-methylcytosine. High resolution mass spectrometry was used to establish the structure of a new modified deoxyribonucleoside from bacteriophage Mu DNA. The modification elaborated by the Mu mom gene converts deoxyadenosine to N-(9-(beta)-D-2'-deoxyribofuranosyl-purin-6-yl)glycinamide. This is the seventh known form of base modification in DNA, and the first characterization of a hypermodified purine.