From microscopic bacteria to the giant whale, every single living organism on Earth uses the same language of life: DNA. Deoxyribonucleoside triphosphates––dNTPs (dATP, dTTP, dGTP, and dCTP)––are the building blocks of DNA and are therefore the “alphabet of life”. A balanced supply of dNTPs is essential for integral DNA transactions such as faithful genome duplication and repair. The enzyme ribonucleotide reductase (RNR) not only synthesizes all four dNTPs but also primarily maintains the crucial individual concentration of each dNTP in a cell. In this thesis we investigated what happens if the crucial balanced supply of dNTPs is disrupted, addressing whether a cell has a mechanism to detect imbalanced dNTP pools and whether all pool imbalances are equally mutagenic. To address these questions, we introduced single amino acid substitutions into loop 2 of the allosteric specificity site of Saccharomyces cerevisiae RNR and obtained a collection of yeast strains with different but defined dNTP pool imbalances. These results directly confirmed that the loop 2 is the structural link between the substrate specificity and effector binding sites of RNR. We were surprised to observe that mutagenesis was enhanced even in a strain with mildly imbalanced dNTP pools, despite the availability of the two major replication error correction mechanisms: proofreading and mismatch repair. However, the mutagenic potential of different dNTP pool imbalances did not directly correlate with their severity, and the locations of the mutations in a strain with elevated dTTP and dCTP were completely different from those in a strain with elevated dATP and dGTP. We then investigated, whether dNTP pool imbalances interfere with cell cycle progression and if they are detected by the S-phase checkpoint, a genome surveillance mechanism activated in response to DNA damage or replication blocks. The S-phase checkpoint was activated by the depletion of one or more dNTPs. In contrast, when none of the dNTP pools was limiting for DNA replication, even extreme and mutagenic dNTP pool imbalances did not activate the S-phase checkpoint and did not interfere with the cell cycle progression. We also observed an interesting mutational strand bias in one of the mutant rnr1 strains suggesting that the S-phase checkpoint may selectively prevent formation of replication errors during leading strand replication. We further used these strains to study the mechanisms by which dNTP pool imbalances induce genome instability. In addition, we discovered that a high dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions, which are difficult to bypass at normal dNTP concentrations. Our results broaden the role of dNTPs beyond ‘dNTPs as the building blocks’ and suggest that dNTPs are not only the building blocks of DNA but also that their concentrations in a cell have regulatory implications for maintaining genomic integrity. This is important as all cancers arise as a result of some kind of genomic abnormality.

A prerequisite for a multicellular organism to survive is the ability to correctly replicate and repair DNA while minimizing the number of heritable mutations. To achieve this, cells need a balanced supply of deoxyribonucleoside triphosphates (dNTPs), the precursors for DNA synthesis. The rate-limiting step in de novo biosynthesis of dNTPs is catalyzed by the enzyme ribonucleotide reductase (RNR). The classic eukaryotic RNR enzyme consists of a large and a small subunit. Together, these subunits form a heterotetrameric RNR complex. The larger subunit harbours active sites whereas the smaller subunit contains a stable tyrosyl free radical. Both subunits are required for RNR activity. Since failure to correctly regulate de novo dNTP biosynthesis can lead to misincorporation of nucleotides into DNA, genetic abnormalities and cell death, RNR activity is tightly regulated. The regulation of RNR activity involves cell cycle-specific expression and degradation of the RNR proteins, as well as binding of allosteric effectors to the large RNR subunit. In this thesis, in vitro assays based on purified recombinant RNR proteins, in combination with in vivo assays, have been used successfully to study the regulation of RNR activity in response to DNA damage. I present new findings regarding the function of an alternative mammalian RNR small subunit, and on the role of a small RNR inhibitor protein of fission yeast, during normal growth and after DNA damage. I also show conclusively that there are fundamental differences in the regulation of dNTP biosynthesis between the cells of higher and lower eukaryotes after DNA damage.

In order to build any structure, a good supply of materials, accurate workers and quality control are needed. This is even the case when constructing DNA, the so-called “Code of Life.” For a species to continue to exist, this DNA code must be copied with incredibly high accuracy when each and every cell replicates. In fact, just one mistake in the 12 million bases that comprise the genome of budding yeast, Saccharomyces cerevisiae, can be fatal. DNA is composed of a double strand helix made up of just four different bases repeated millions of times. The building blocks of DNA are the deoxyribonucleotides (dNTPs); dCTP, dTTP, dATP and dGTP. Their production and balance are carefully controlled within each cell, largely by the key enzyme Ribonucleotide Reductase (RNR). Here, we studied how the enzymes that copy DNA, the replicative polymerases α, δ and ε, cope with the effects of an altered dNTP pool balance. An introduced mutation in the allosteric specificity site of RNR in a strain of S. cerevisiae, rnr1-Y285A, leads to elevated dCTP and dTTP levels and has been shown to have a 14-fold increase in mutation rate compared to wild type. To ascertain the full effects of the dNTP pool imbalance upon the replicative polymerases, we disabled one of the major quality control systems in a cell that corrects replication errors, the post-replicative Mismatch Repair system. Using both the CAN1 reporter assay and whole genome sequencing, we found that, despite inherent differences between the polymerases, their replication fidelity was affected very similarly by this dNTP pool imbalance. Hence, the high dCTP and dTTP forced Pol ε and Pol α/δ to make the same mistakes. In addition, the mismatch repair machinery was found to correct replication errors driven by this dNTP pool imbalance with highly variable efficiencies. Another mechanism to protect cells from DNA damage during replication is a checkpoint that can be activated to delay the cell cycle and activate repair mechanisms. In yeast, Mec1 and Rad53 (human ATR and Chk1/Chk2) are two key S-phase checkpoint proteins. They are essential as they are also required for normal DNA replication and dNTP pool regulation. However the reason why they are essential is not well understood. We investigated this by mutating RAD53 and analyzing dNTP pools and gene interactions. We show that Rad53 is essential in S-phase due to its role in regulating basal dNTP levels by action in the Dun1 pathway that regulates RNR and Rad53’s compensatory kinase function if dNTP levels are perturbed. In conclusion we present further evidence of the importance of dNTP pools in the maintenance of genome integrity and shed more light on the complex regulation of dNTP levels.