Abstract
Activation-induced deaminase (AID) and apolipoprotein B mRNA-editing enzyme catalytic subunit (APOBEC) enzymes convert cytosines to uracils, creating signature mutations that have been used to predict sites targeted by these enzymes. Mutation-based targeting maps are distorted by the error-prone or error-free repair of these uracils and by selection pressures. To directly map uracils created by AID/APOBEC enzymes, here we used uracil-DNA glycosylase and an alkoxyamine to covalently tag and sequence uracil-containing DNA fragments (UPD-Seq). We applied this technique to the genome of repair-defective, APOBEC3A-expressing bacterial cells and created a uracilation genome map, i.e. uracilome. The peak uracilated regions were in the 5'-ends of genes and operons mainly containing tRNA genes and a few protein-coding genes. We validated these findings through deep sequencing of pulldown regions and whole-genome sequencing of independent clones. The peaks were not correlated with high transcription rates or stable RNA:DNA hybrid formation. We defined the uracilation index (UI) as the frequency of occurrence of TT in UPD-Seq reads at different original TC dinucleotides. Genome-wide UI calculation confirmed that APOBEC3A modifies cytosines in the lagging-strand template during replication and in short hairpin loops. APOBEC3A's preference for tRNA genes was observed previously in yeast, and an analysis of human tumor sequences revealed that in tumors with a high percentage of APOBEC3 signature mutations, the frequency of tRNA gene mutations was much higher than in the rest of the genome. These results identify multiple causes underlying selection of cytosines by APOBEC3A for deamination, and demonstrate the utility of UPD-Seq.
Highlights
Activation-induced deaminase (AID) and apolipoprotein B mRNA-editing enzyme catalytic subunit (APOBEC) enzymes convert cytosines to uracils, creating signature mutations that have been used to predict sites targeted by these enzymes
The third pathway for the acquisition of uracils in DNA is through the action of the AID/APOBEC3 family of ssDNAspecific cytosine deaminases found in most vertebrates (10 –13)
This technology involves the use of a disulfide link-containing chemical, ssARP [34] (Fig. 1A), to tag abasic sites created by the removal of uracils from DNA
Summary
We created a new method for pulldown of uracilated DNA fragments for sequencing (uracil pulldown sequencing; UPDseq). As the E. coli cells were grown in rich medium, they should contain multiple replication forks [40] and this is the likely explanation for the higher density of uracil pulldown fragments near the origin of replication compared with other parts of the genome Together, these results show that the uracil pulldown and sequencing strategy outlined in Fig. 1B works despite the chemical scar left behind by ssARP in DNA. The uracilation seen in cells lacking an active A3A is likely due to water-mediated cytosine deaminations, and has been described previously to cause a replicative strand bias in C to T mutations [43] These results show that the UI index is useful for detecting DNA strand preferences of A3A, without a need to isolate and sequence individual mutants. This suggests that the regions represented by the peaks accumulated uracils created by A3A or water much more frequently than other genomic regions and this is why they are over-represented in the pulldown libraries
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