**4.1 Modifications to uracil**

Of all the bases, U is the most modified in RNA [32, 33], while in DNA, U has been reported to undergo modifications including methylation [32], hydroxymethylation [34] and oxidation [35]. Methylation, the addition of a methyl group to the nitrogen atom in the ring structure of uracil, can occur at several sites on the base, including the N1 and N3 positions [32]. Hydroxymethylation, the addition of a hydroxymethyl group to the N3 position, is a common modification [32, 35]. Oxidation can also lead to the formation of several different uracil derivates, such as 5-hydroxymethyluracil and 5-formyluracil [35]. Other less prevalent modifications may include glutathionylation, nitration or phosphorylation [36]. These modifications can affect the stability and function of the DNA molecule and may also play a role in regulating gene expression and the development of diseases such as cancer [37, 38].

## **4.2 Maintenance of uracil-free DNA uracil**

Hydrolytic depurination produces about 10,000 abasic sites per cell per day, while hydrolytic deamination of cytosines produces 70–200 uracil bases per day in DNA [13]. The inappropriate U is recognized by family of human UDGs, which cleaves the N-glycosidic bond and thereby generates an abasic sites in the DNA, which are themselves cytotoxic and potentially mutagenic [39]. As mentioned in Section 2, U can arise in DNA either by C deamination or by U mis-incorporation. Deamination of C to U is always mutagenic, if not corrected, as U:G mismatches always leads to C→T or G→A transitions during DNA synthesis and is, interestingly, the most frequent spontaneous mutation of cells, and often found in human tumours. On the other hand, misincorporation of U should not lead to a mutation during DNA synthesis, since U:A pairs would lead to T:A pairs; however, DNA repair mechanisms can be error-prone and, therefore, it is best for the cell to avoid U DNA misincorporation from the onset, as discussed in Section 2.

In almost all the organisms, the nucleotide pools are essential for the correct DNA replication and U is one of the most frequently occurring error bases in DNA; different strategies for "keeping free" the organism of unbalanced levels of nucleotides exists [22]. In this direction, four superfamilies of NTP (nucleoside triphosphate) pyrophosphatases including the nudix hydrolases, trimeric dUTPase, inosine triphosphate pyrophosphatases (ITPases) and all α NTP pyrophosphatases function to hydrolyze the α-β phosphodiester bond of (d)NTPs to monophosphate and pyrophosphoric acid (PPi) focussing on the non-canonical NTPs [22, 23]. The deoxycytidine triphosphatase (dCTPase) and dUTPase are the two main nucleotide hydrolases involved in the elimination of non-canonical nucleotides [22, 24].

The most important mechanisms to maintain U-free-DNA are dUTPase and UDGs [40–42].
