Characterizing DNA photo-oxidation reactions by high-resolution mass measurements with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

High-resolution mass measurements by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were employed to characterize laser-induced oxidation of guanine in a small synthetic deoxyoligonucleotide. The oligonucleotide was exposed to high-intensity UV radiation at 266 nm to produce modifications on the guanine base. The primary product showed a +16 Da mass shift relative to the original strand, whereas secondary products showed mass shifts of +32 and +34 Da. The mass shift of the primary product is consistent with an 8-oxoguanine modification. However, the reactivity of the primary product with hot piperidine and other secondary oxidizing agents was different from that of a synthetic oligonucleotide containing 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxoG). Based upon the results, a new reaction scheme involving the formation of an epoxide ring across the C-4 and C-5 positions by UV laser-induced oxidation is suggested. The results also illustrate the ability of MALDI to characterize chemical reactivity rapidly at the a low picomolar level.

Identification of single stranded regions of DNA by enzymatic digestion with matrix-assisted laser desorption/ionization analysis.

Elucidating structure function relationships of DNA in cellular processes requires fast, reliable methods that can be applied to picomole amounts of sample. Higher order structure can be inferred by distinguishing paired and unpaired regions. It is shown here that enzymatic digestion coupled with product analysis by matrix-assisted laser desorption ionization (MALDI) is able to identify unpaired bases within structured DNA regions. The method is demonstrated with DNA duplexes having a five nucleotide mismatch as a 5' overhang, a 3' overhang, and an internal loop. Exo- and endonuclease digestions are performed under solution conditions (temperature, annealing, and enzyme buffers) which promote base pairing and specific enzyme activity. For each type of mismatch, the length and sequence of the single stranded region can be inferred from MALDI spectra taken as a function of digestion time.

RNA structure inhibits the TRAP (trp RNA-binding attenuation protein)-RNA interaction.

TRAP (trp RNA-binding attenuation protein) regulates expression of the tryptophan biosynthetic genes in response to tryptophan in Bacillus subtilis by binding to two sites containing a series of 9 or 11 (G/U)AG triplet repeats that are generally separated by two or three spacer nucleotides. Previous mutagenesis experiments have identified three TRAP residues, Lys-37, Lys-56, and Arg-58 that are essential for RNA binding. The location of these residues on the TRAP oligomer supports the proposal that RNA binds TRAP by encircling the TRAP oligomer. In this work, we show that RNAs containing 11 GAG or UAG repeats separated by CC dinucleotide spacers (((G/U)AGCC)11) form stable structures that inhibit binding to TRAP. This conclusion is based on the effects of temperature and Mg2+ on the affinity of TRAP for RNAs with CC spacers combined with UV hyperchromicity and circular dichroism. Furthermore, introducing the base analogue 7-deazaguanosine in the ((G/U)AGCC)11 RNAs stabilized the TRAP-RNA interaction. This effect was associated with decreased stability of the RNA structure as measured by circular dichroism spectroscopy. The precise nature of the structure of the ((G/U)AGCC)11 RNAs is not known but evidence is presented that it involves noncanonical interactions. We also observed that substitution of Arg-58 with Lys further reduced the ability of TRAP to interact with structured RNAs. Since in vivo function of TRAP may involve binding to structured RNAs, we suggest a potential function for this residue, which is conserved in TRAP from three different bacilli.

An unexpected pathway for the metabolic degradation of 1,3-dialkyl-3-acyltriazenes.

In the presence of NADPH, rat liver microsomes catalyzed the degradation of a series of 1,3-dialkyl-3-acyltriazenes, and the extent of the reaction was correlated with compound lipophilicity. In the case of two methylcarbamoyltriazenes, 1-(2-chloroethyl)-3-benzyl-3- (methylcarbamoyl)triazene (CBzM) and 1-(2-chloroethyl)-3-methyl-3-(methylcarbamoyl)triazene (CMM), microsomal metabolites were isolated. Identification of the CBzM metabolites as 1-(2-chloroethyl)-3-benzyl-3-(hydroxymethylcarbamoyl)triazene and 1-(2-chloroethyl-3-benzyl-3-carbamoyltriazine, and the CMM metabolite as 1-(2-chloroethyl)-3-methyl-3-(hydroxymethylcarbamoyl)triazene indicated that the first metabolic step involves hydroxylation of the methylcarbamoyl substituent. Detailed studies of the metabolism of CBzM indicated that the Km for the reaction was 84 microM, and that metabolism was more efficient if microsomes were prepared from male than from female rats. During prolonged incubation, the metabolites of CBzM were also degraded. The degradation of CBzM and its metabolites was inhibited by SKF-525A and metyrapone, suggesting the involvement of a cytochrome P450 isozyme, and supporting the hypothesis that the process is oxidative rather than hydrolytic in both cases. Metabolic oxidation represents an alternative pathway to chemical or enzymatic hydrolysis for the in vivo decomposition of (methylcarbamoyl)triazenes. This mechanism may ultimately explain the antitumor efficacy and low acute toxicity of selected compounds.