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Additional Data for Intrinsic strand-incision activity of human UNG predicts nick generation in immunoglobulin gene diversification

Uracil arises in DNA by hydrolytic deamination of cytosine (C) and by erroneous incorporation of deoxyuridine monophosphate opposite adenine, where the former event is devastating by generation of C → thymine transitions. The base excision repair (BER) pathway replaces uracil by the correct base. In human cells two uracil-DNA glycosylases (UDGs) initiate BER by excising uracil from DNA; one is hSMUG1 (human single-strand-selective mono-functional UDG). We report that repair initiation by hSMUG1 involves strand incision at the uracil site resulting in a 3′-α,β-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5′-phosphate. UIP is removed from the 3′-end by human apurinic/apyrimidinic (AP) endonuclease 1 preparing for single-nucleotide insertion. hSMUG1 also incises DNA or processes UIP to a 3′-phosphate designated uracil-DNA processing product (UPP). UIP and UPP were indirectly identified and quantified by polyacrylamide gel electrophoresis and chemically characterised by matrix-assisted laser desorption/ionisation time-of-flight mass-spectrometric analysis of DNA from enzyme reactions using 18O- or 16O-water. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2′ occurs via the formation of a C1′ enolate intermediate. A three-phase kinetic model explains rapid uracil excision in phase 1, slow unspecific enzyme adsorption/desorption to DNA in phase 2 and enzyme-dependent AP site incision in phase 3.

tRNA is the most heavily modified of all RNA types, with typically 10-20% of the residues being post-transcriptionally altered. Unravelling the modification pattern of a tRNA is a challenging task; there are 92 currently known tRNA modifications [1], many of which are chemically similar. Furthermore, the tRNA has to be investigated with single-nucleotide resolution in order to ensure complete mapping of all modifications. In the present work, we characterized tRNA(Lys) (UUU) from Trypanosoma brucei, and provide a complete overview of its post-transcriptional modifications. The first step was MALDI-TOF MS of two independent digests of the tRNA, with RNase A and RNase T1, respectively. This revealed digestion products harbouring mass-changing modifications. Next, the modifications were mapped at the nucleotide level in the RNase products by tandem MS. Comparison with the sequence of the unmodified tRNA revealed the modified residues. The modifications were further characterized at the nucleoside level by chromatographic retention time and fragmentation pattern upon higher-order tandem MS. Phylogenetic comparison with modifications in tRNA(Lys) from other organisms was used through the entire analysis. We identified modifications on 12 nucleosides in tRNA(Lys) (UUU), where U47 exhibited a novel modification, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, based on identical chromatographic retention and MS fragmentation as the synthetic nucleoside. A37 was observed in two versions: a minor fraction with the previously described 2-methylthio-N(6) -threonylcarbamoyl-modification, and a major fraction with A37 being modified by a 294.0-Da moiety. The latter product is the largest adenosine modification reported so far, and we discuss its nature and origin.

Posttranscriptional modifications on the base or sugar of ribonucleosides generally result in mass increases that can be measured by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a direct and accurate means of determining the masses of RNAs. Mass spectra produced by MALDI are relatively straightforward to interpret, because they are dominated by singly charged ions, making it possible to analyze complex mixtures of RNA oligonucleotides ranging from trinucleotides up to 20-mers. Analysis of modifications within much longer RNAs, such as ribosomal RNAs, can be achieved by digesting the RNA with nucleotide-specific enzymes. In some cases, it may be desirable to isolate specific sequence regions before MALDI-MS analysis, and this requires a few additional steps. The method is applicable to the study of modified RNAs from cell extracts as well as RNA modifications added in cell-free in vitro systems. MALDI-MS is particularly useful in cases in which other techniques such as those involving primer extension or chromatographic analyses are not practicable. To date, MALDI-MS has been used to localize rRNA modifications that are involved in fundamental processes in protein synthesis as well as methylations that confer resistance to antibiotics. For several rRNA sites, MALDI-MS has served an essential role in the identification of the enzymes that catalyze the modifications.