Mechanisms of dNTP supply that play an essential role in maintaining genome integrity in eukaryotic cells

Abstract

Optimization of intracellular concentrations of dNTPs is critical for the fidelity of DNA synthesis during DNA replication and repair because levels that are too high or too low can easily lead to increased rates of mutagenesis. Recent advances in the analysis of intracellular concentrations of dNTPs have suggested that eukaryotes use diverse mechanisms in supplying dNTPs for DNA synthesis during DNA replication and repair. The enzyme ribonucleotide reductase (RNR) is a key enzyme involved in the synthesis of dNTPs. We found that Tip60-dependent recruitment of RNR at sites of DNA damage is essential for supplying a sufficient amount of dNTPs for mammalian DNA repair. In this review, we focus on recent findings related to RNR regulation in eukaryotes of the dNTPs supplied for DNA synthesis. We also discuss the effect of this regulation on mutagenesis and tumorigenesis.

Figure 1

Ribonucleotide reductase (RNR) and its allosteric regulation. (A) The enzyme RNR catalyzes the de novo synthesis of dNTPs. Catalysis of ribonucleoside 5′‐diphosphate (NDP) involves a reduction at the 2′‐carbon of ribose 5‐phosphate to form the 2′‐deoxy derivative‐reduced 2′‐deoxyribonucleoside 5′‐diphosphate (dNDP). This reduction is initiated with the generation of a free radical. Following a single reduction, RNR requires electrons donated from the dithiol groups of the protein thioredoxin. Regeneration of thioredoxin occurs when nicotinamide adenine dinucleotide phosphate (NADPH) provides two hydrogen atoms that are used to reduce the disulfide groups of the thioredoxin protein. (B) Allosteric regulation of RNR. RNR is activated by binding ATP or inactivated by binding dATP to the activity site located on the large subunit Rnr1 (R1). When the enzyme is activated, substrates are reduced if the corresponding effectors bind to the allosteric substrate specificity site as follows. When dATP or ATP is bound at the allosteric site, the enzyme accepts UDP and CDP into the catalytic site. When dGTP is bound, ADP enters the catalytic site. When dTTP is bound, GDP enters the catalytic site. The substrates (UDP, CDP, ADP, and GDP) are converted to dNTPs. R2, small subunit Rnr2‐Rnr4.

Figure 2

Regulation of subcellular localization of ribonucleotide reductase (RNR) in budding yeast. The small subunit Rnr2‐Rnr4 (R2) localizes at the nucleus, whereas the large subunit Rnr1 (R1) is cytoplasmic outside of S phase. After DNA damage or during S phase, the Rnr2‐Rnr4 subunit enters the cytoplasm enabling it to bind to Rnr1, forming an active complex. Dif1 directly binds to the R2 complex which promotes the import of R2 into the nucleus. The imported R2 then forms a complex with Wtm1, which anchors the complex in the nucleus. In the presence of DNA damage, the association of R2 with Wtm1 is disrupted. Furthermore, DNA damage‐induced activation of the Mec1‐Rad53‐Dun1 axis directly phosphorylates (P) Dif1, which inactivates and triggers its degradation. A reduction in Dif1, together with the dissociation of R2 from Wtm1 after DNA damage, allows R2 to enter the cytoplasm.

Figure 3

Sharp contrasts between yeasts and mammals in the regulation of dNTP pools after DNA damage. After DNA damage, whole dNTP pools in mammalian cells are almost unchanged, but those in yeasts are drastically increased.^{( 10 )} These observations suggest that there is a unique mechanism in mammals by which dNTPs are compartmentalized close to the damage sites during the DNA repair process, thereby providing a high local concentration. Black dots represent dNTPs (lower panels).

Figure 4

Mechanism of dNTP supply to DNA damage sites in mammals After DNA damage, the Tip60/ribonucleotide reductase (RNR) complex is rapidly recruited to the damage sites. Tip60 acetylates (Ac) histone H4 surrounding these sites and this acetylation enhances recruitment of repair enzymes and several checkpoint proteins. Recruited RNR can supply sufficient amounts of dNTPs for proper DNA repair.