*4.2.1 dUTPase*

The function of dUTPase is to hydrolyze dUTP to dUMP and pyrophosphate, providing a dUMP precursor for the dTMP synthesis, maintaining the balanced

dUTP/dTTP ratio and ultimately DNA integrity [43]; this reaction facilitates the cells avoidance of dUTP DNA misincorporation by DNA polymerases during replication (**Figure 1**) [44].

Two distinct protein isoforms of dUTPase, one nuclear and the other mitochondrial, in human cells have been reported [45]. Nuclear dUTPase (DUT-N) and mitochondrial dUTPase (DUT-M) are encoded by two distinct mRNA species of 1.1 and 1.4 kilobases respectively, nonetheless, the dUTPase gene (*DUT*) encode both nuclear and mitochondrial isoforms and arise to mature form by splicing process using different exon patterns [46].

In normal cells, the expression of dUTPase varies, where fluctuations in expression are dependent upon the current state of the cell cycle. Stimulation from mitogenic signals to initiate mitosis triggers the cell to progress from the resting G0-phase into G1/S-phase [47]. It is during S-phase of the cell cycle when the DUT-N is increased to provide the dUMP substrate required for dTMP synthesis and subsequent deoxythymidine triphosphate (dTTP) for incorporation into newly synthesized DNA [48]. Overall, the specific activity is over 16,000 nmol of dUMP hydrolyzed per min/mg of dUTPase [49].

#### *4.2.2 Base excision repair*

There are four UDGs present in mammalian cells: UNG, SMUG1, TDG and MBD4 [50] that function to recognize and remove U from DNA. This family of enzymes provides redundancy which may be required for specific circumstances and highlights the importance of this repair process. UNG remains central to the repair of U:A misincorporated uracil, whereas all family members are involved in the U:G repair [29].

The process of uracil repair produces an abasic sites, which forms part of Base Excision Repair (BER) pathway in humans, a process that repairs small, non-helix distorting base lesions in the genome [51]. Briefly, a UDG detects U within DNA and 'flips' it out of the double-helix and cleaves the U leaving an abasic site. An AP-endonuclease (APE) then cleaves the DNA-backbone 5′ of the abasic site, creating a single-strand break [52]. During short-patch BER, DNA polymerase β (POLβ) inserts the correct nucleotide into the abasic site and has lyase activity that removes the deoxyribosephosphate (dRP) left over from the abasic site [53]. The open 3′ end (that is left over after DNA polymerase activity) can then be sealed by DNA ligase III (LIG3) and its co-factor XRCC1, removing what is a single-strand break [54, 55]. Alternatively, during long-patch BER; POLδ, POLε and PCNA inserts multiple nucleotides from the abasic site, displacing the downstream DNA (which also contains dRP at its 5′ end), creating a flap [53]. The flap endonuclease 1 (FEN1) then removes this 5′ flap of DNA [56] and DNA ligase 1 (LIG1) is able to seal the single-strand break in the DNA backbone left after this process [57]. See **Figure 2** for BER schematic.
