In yeast, dNTP pools expand drastically during DNA damage response. We show that similar dNTP elevation occurs in strains, in which intrinsic replisome defects promote the participation of error-prone DNA polymerase ζ (Polζ) in replication of undamaged DNA. To understand the significance of dNTP pools increase for Polζ function, we studied the activity and fidelity of four-subunit Polζ (Polζ4) and Polζ4-Rev1 (Polζ5) complexes in vitro at 'normal S-phase' and 'damage-response' dNTP concentrations. The presence of Rev1 inhibited the activity of Polζ and greatly increased the rate of all three 'X-dCTP' mispairs, which Polζ4 alone made extremely inefficiently. Both Polζ4 and Polζ5 were most promiscuous at G nucleotides and frequently generated multiple closely spaced sequence changes. Surprisingly, the shift from 'S-phase' to 'damage-response' dNTP levels only minimally affected the activity, fidelity and error specificity of Polζ complexes. Moreover, Polζ-dependent mutagenesis triggered by replisome defects or UV irradiation in vivo was not decreased when dNTP synthesis was suppressed by hydroxyurea, indicating that Polζ function does not require high dNTP levels. The results support a model wherein dNTP elevation is needed to facilitate non-mutagenic tolerance pathways, while Polζ synthesis represents a unique mechanism of rescuing stalled replication when dNTP supply is low.
Even small variations in dNTP concentrations decrease DNA replication fidelity, and this observation prompted us to analyze genomic cancer data for mutations in enzymes involved in dNTP metabolism. We found that sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), a deoxyribonucleoside triphosphate triphosphohydrolase that decreases dNTP pools, is frequently mutated in colon cancers, that these mutations negatively affect SAMHD1 activity, and that severalSAMHD1mutations are found in tumors with defective mismatch repair. We show that minor changes in dNTP pools in combination with inactivated mismatch repair dramatically increase mutation rates. Determination of dNTP pools in mouse embryos revealed that inactivation of oneSAMHD1allele is sufficient to elevate dNTP pools. These observations suggest that heterozygous cancer-associatedSAMHD1mutations increase mutation rates in cancer cells.
The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues.
Defects in deoxyribonucleoside triphosphate (dNTP) metabolism are associated with a number of mitochondrial DNA (mtDNA) depletion syndromes (MDS). These disorders affect the muscles, liver, and brain, and the concentrations of dNTPs in these tissues are already normally low and are, therefore, difficult to measure. Thus, information about the concentrations of dNTPs in tissues of healthy animals and animals with MDS are important for mechanistic studies of mtDNA replication, analysis of disease progression, and the development of therapeutic interventions. Here, we present a sensitive method for the simultaneous analysis of all four dNTPs as well as all four ribonucleoside triphosphates (NTPs) in mouse muscles using hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry. The simultaneous detection of NTPs allows them to be used as internal standards for the normalization of dNTP concentrations. The method can be applied for measuring dNTP and NTP pools in other tissues and organisms.
Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac abnormalities and impaired cardiac conduction systems. As a result, they died between two and four weeks after birth. Taken together, our studies provide the first empirical evidence that both the de novo and salvage pathways are essential to keeping the dNTP concentration at an optimal range to prevent mutagenesis, carcinogenesis, and mortality.
The high accuracy of DNA replication is achieved through the nucleotide selectivity of DNA polymerases, polymerase proofreading, and the mismatch repair (MMR) system that act in series. While defects in proofreading and MMR are strongly associated with the development of cancers, decreased nucleotide selectivity due to mutations in replicative DNA polymerases is an uncommon driver of cancer development. Because nucleotide selectivity can also be decreased by imbalanced dNTP pools, we investigated to what extent imbalanced dNTP pools can induce cancers. To this end we developed a mouse model with a mutation in the allosteric specificity site of ribonucleotide reductase, which is responsible for the balanced production of dNTPs. These mice had ~2-fold increased dCTP and dTTP levels and normal dATP and dGTP levels. Despite this mild dNTP pool imbalance, mutant mice had a higher incidence and an earlier onset of cancers, and these were different from the cancers that developed in wild-type controls. Because dNTP pool imbalances can be caused by defects in a plethora of genes, we propose that decreased nucleotide selectivity might be a major factor contributing to the development of spontaneous cancers.
The building blocks of DNA, dNTPs, can be produced de novo or can be salvaged from deoxyribonucleosides. However, to what extent the absence of de novo dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated de novo dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the de novo production of dNTPs, at embryonic day 13. All other tissues had normal de novo dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in wild-type postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for de novo dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in wildtype hearts.
Ribonucleotides (rNMPs) incorporated in the nuclear genome are a well-established threat to genome stability and can result in DNA strand breaks when not removed in a timely manner. However, the presence of a certain level of rNMPs is tolerated in mitochondrial DNA (mtDNA) although aberrant mtDNA rNMP content has been identified in disease models. We investigated the effect of incorporated rNMPs on mtDNA stability over the mouse life span and found that the mtDNA rNMP content increased during early life. The rNMP content of mtDNA varied greatly across different tissues and was defined by the rNTP/dNTP ratio of the tissue. Accordingly, mtDNA rNMPs were nearly absent in SAMHD1 -/- mice that have increased dNTP pools. The near absence of rNMPs did not, however, appreciably affect mtDNA copy number or the levels of mtDNA molecules with deletions or strand breaks in aged animals near the end of their life span. The physiological rNMP load therefore does not contribute to the progressive loss of mtDNA quality that occurs as mice age.