Abstract
Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1−/− mice. We found that 5-hydroxymethyluracil accumulated in Smug1−/− tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1−/− mice did not accumulate uracil in their genome and Ung−/− mice showed slightly elevated uracil levels. Contrastingly, Ung−/−Smug1−/− mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.
Highlights
Proliferating cells, with an estimated ~104 dUTPs misincorporated per genome per cell-division cycle[1, 2]
Uracil is primarily excised by uracil-DNA glycosylases (UDGs)[6], and the two UDGs capable of repairing both U:A and U:G from nuclear DNA are uracil-DNA glycosylase (UNG)[7] and single-strand selective monofunctional uracil-DNA glycosylase 1 (SMUG1)[8]
We measured the levels of UNG and SMUG1 substrates in genomic DNA of mice deficient in one or both enzymes to clarify their relative importance in base excision repair (BER)
Summary
Proliferating cells, with an estimated ~104 dUTPs misincorporated per genome per cell-division cycle[1, 2]. The mutagenic potential of hmU depends on its origin: when present in a hmU:A base pair after direct oxidation of thymidine it is likely innocuous[24], but hmU has been suggested to form as an intermediate of oxidative demethylation of 5-methylcytosine[25], leading to a premutagenic hmU:G mispair These substrates are not shared between SMUG1 and UNG, which could explain the additive mutagenic effect of suppressing SMUG1 expression in Ung−/− MEFs. there are several unresolved questions regarding the distinct functions of UNG and SMUG1 in in vivo uracil repair and their importance for limiting spontaneous mutagenesis. Ung−/−Smug1−/− mice had more than an additive increase in uracil content compared to either single knockout This shows that SMUG1 is important for uracil repair in cells with functional UNG. The two enzymes complement each other during the repair of uracil in vivo and the mutagenic potential of defective uracil repair is best assessed in cells lacking both enzymes: whole genome sequencing of UNG/SMUG1-deficient tumours revealed that the loss of uracil repair by UNG and SMUG1 primarily leads to C to T transition mutagenesis at CpG dinucleotides
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