**3. Biosynthesis of NTPs**

Nucleotide synthesis *begins with U*(ridine monophosphate) and *I*(nosine monophosphate) [22–24]*.* The success of DNA replication is dependent upon a number of factors including the availability of balanced nucleotide pools and the cellular ability to recognise misincorporated bases in DNA and initiate their removal and subsequent repair. In the quiescent state, the genomic content of the cell is at its' minimum. Following stimulation to enter the cell cycle and initiate S-phase, DNA biosynthesis is up-regulated and therefore access to the required deoxynucleotide triphosphates (dNTPs) is vital for errorless replication of the genetic material [25]. The enzyme ribonucleotide reductase (RNR) is the initiating factor that induces the reduction of ribonucleoside diphosphates into their respective deoxyribonucleoside diphosphates (dNDPs) [26, 27]. Additional phosphorylation of the nucleosides occurs through the action of nucleoside diphosphate kinase (NDPK), converting the dNDP's to dNTP. However, the synthesis of dTTP requires additional steps within the biosynthesis pathway (**Figure 1**; for an in-depth review, see ref. [28]).

#### **Figure 1.**

*Uracil biosynthesis and incorporation into genomic RNA and DNA. A simplified schematic showing how uracil (U, purple) and thymidine (T, red) are incorporated into DNA. Normally, uracil is metabolised to UDP then UTP, allowing it to be incorporated into RNA. UDP can also be processed by ribonucleotide reductase (RNR, composed of RRM1/2/2B subunits) to dUDP, which is further metabolised to dUTP. Cells have evolved a system where dUTP levels are kept low by dUTPase converting it into dUMP, which is a substrate for dTTP production. This maintains a high cellular dTTP:dUTP ratio, ensuring minimal uracil misincorporation into DNA since DNA polymerase has similar affinities for dTTP and dUTP. Arrows indicate the direction of a metabolic reaction by an enzyme, double ended arrows indicate a single enzyme can perform a metabolic reaction in both directions. Abbreviations: U = uracil, rU = uridine, dU = deoxyuridine, (d)UMP = (deoxy)uridine monophosphate, (d)UDP = (deoxy)uridine diphosphate, (d)UTP = (deoxy)uridine triphosphate, dT = deoxythymidine, (d) TMP = (deoxy)thymidine monophosphate, (d)TDP = (deoxy)thymidine diphosphate, (d)TTP = (deoxy) thymidine triphosphate, CH2-FH4 = 5,10-methylenetetrahydrofolate, FH2 = dihydrofolate, DNA Polβ = DNA polymerase β, RNR = ribonucleotide reductase, and dUTPase = dUTP nucleotidohydrolase.*
