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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.

To transfer the information in the genome from mother cell to daughter cell, the DNA replication must be carried out only once and with very high fidelity prior to every cell division. In yeast there are several different DNA polymerases involved in DNA replication and/or DNA repair. The two replicative DNA polymerases, DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon), which both include a proofreading 3´→5´exonuclease activity, can replicate and proofread the genome with a very high degree of accuracy. The aim of this thesis was to gain a better understanding of how the enigmatic DNA polymerase epsilon participates in DNA transactions.

To investigate whether Pol epsilon or Pol delta is responsible for the synthesis of DNA on the lagging strand, the processing and assembly of Okazaki fragments was studied. Pol delta was found to have a unique property called “idling” which, together with the flap-endonuclease (FEN1), maintained a ligatable nick for DNA ligase I. In contrast, Pol epsilon was found to lack the ability to “idle” and interact functionally with FEN-1, indicating that Pol epsilon is not involved in processing Okazaki fragments. Together with previous genetic studies, it was concluded that Pol delta is the preferred lagging strand polymerase, leaving Pol epsilon to carry out some other function.

The structure of Pol epsilon was determined by cryo-electron microscopy, to a resolution of ~20 Å. Pol epsilon is composed of a globular “head” domain consisting of the large catalytic subunit Pol2p, and a “tail” domain, consisting of the small subunits Dpb2p, Dpb3p, and Dpb4p. The two separable domains were found to be connected by a flexible hinge. Interestingly, the high intrinsic processivity of Pol epsilon depends on the interaction between the tail domain and double-stranded DNA.

As a replicative DNA polymerase, Pol epsilon encounters different lesions in DNA. It was shown that Pol epsilon can perform translesion synthesis (TLS) through a model abasic site in the absence of external processivity clamps under single-hit conditions. The lesion bypass was dependent of the sequence on the template and also on a proper interaction of the “tail”domain with the primer-template.

Yeast cells treated with a DNA damaging agent and devoid of all TLS polymerases showed improved survival rates in the presence of elevated levels of dNTPs. These genetic results suggested that replicative polymerases may be engaged in the bypass of some DNA lesions. In vitro, Pol epsilon was found to bypass 8-OxoG at elevated dNTP levels. Together, the in vitro and in vivo results suggest that the replicative polymerases may be engaged in bypass of less bulky DNA lesions at elevated dNTP levels.

In conclusion, the low-resolution structure presented represents the first structural characterization of a eukaryotic multi-subunit DNA polymerase. The replicative DNA polymerase Pol epsilon can perform translesion synthesis due to an interaction between the tail domain and double-stranded DNA. Pol epsilon may also bypass less bulky DNA lesions when there are elevated dNTP concentrations in vivo.

All living organisms are made of cells, within which genetic information is stored on long strands of deoxyribonucleic acid (DNA). The DNA encodes thousands of different genes and provides the blueprint for all of the structures and activities occurring within the cell. The building blocks of DNA are the four deoxyribonucleotides, dATP, dGTP, dTTP, and dCTP, which are collectively referred to as dNTPs.

The key enzyme in the production of dNTPs is ribonucleotide reductase (RNR). In the budding yeast Saccharomyces cerevisiae, the concentrations of the individual dNTPs are not equal and it is primarily RNR that maintains this balance. Maintenance of the dNTP pool balance is critical for accurate DNA replication and DNA repair since elevated and/or imbalanced dNTP concentrations increase the mutation rate and can ultimately lead to genomic instability and cancer. In response to DNA damage, the overall dNTP concentration in S. cerevisiae increases. Cell survival rates increase as a result of the elevated concentration of dNTPs, but the cells also suffer from a concomitant increase in mutation rates. When the replication machinery encounters DNA damage that it cannot bypass, the replication fork stalls and recruits specialized translesion synthesis (TLS) polymerases that bypass the damage so that replication can continue. We hypothesized that elevated dNTP levels in response to DNA damage may allow the TLS polymerases to more efficiently bypass DNA damage. To explore this possibility, we deleted all known TLS polymerases in a yeast strain in which we could artificially increase the dNTP concentrations. Surprisingly, even though all TLS polymerases had been deleted, elevated dNTP concentrations led to increased cell survival after DNA damage. These results suggest that replicative DNA polymerases may be involved in the bypass of certain DNA lesions under conditions of elevated dNTPs. We confirmed this hypothesis in vitro by demonstrating that high dNTP concentrations result in an increased efficiency in the bypass of certain DNA lesions by DNA polymerase epsilon, a replicative DNA polymerase not normally associated with TLS activity.

We asked ourselves if it would be possible to create yeast strains with imbalanced dNTP concentrations in vivo, and, if so, would these imbalances be recognized by the checkpoint control mechanisms in the cell. To address these questions, we focused on the highly conserved loop2 of the allosteric specificity site of yeast Rnr1p. We introduced several mutations into RNR1-loop2 that resulted in changes in the amino acid sequence of the protein.

Each of the rnr1-loop2 mutation strains obtained had different levels of individual dNTPs relative to the others. Interestingly, all of the imbalanced dNTP concentrations led to increased mutation rates, but these mutagenic imbalances did not activate the S-phase checkpoint unless one or several dNTPs were present at concentrations that were too low to sustain DNA replication. We were able to use these mutant yeast strains to successfully correlate amino acid substitutions within loop2 of Rnr1p to specific ratios of dNTP concentrations in the cells. We also demonstrated that specific imbalances between the individual dNTP levels result in unique mutation spectra. These mutation spectra suggest that the mutagenesis that results from imbalanced dNTP pools is due to a decrease in fidelity of the replicative DNA polymerases at specific DNA sequences where they are more likely to make a mistake. The mutant rnr1-loop2 strains that we have created with defined dNTP pool imbalances will be of great value for in vivo studies of polymerase fidelity, translesion synthesis by specialized DNA polymerases, and lesion recognition by the DNA repair machinery.