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Disruptions in deoxynucleoside triphosphate (dNTP) supply impair DNA replication and lead to genomic instability. While exogenous ribonucleosides (rNuc) have been suggested to alleviate replication stress by increasing dNTP levels, their precise metabolic effects remain unclear. Here, we show that rNuc supplementation primarily elevates CTP and UTP levels, with only modest increases in dCTP, and has minimal impact on replication fork speed across multiple mammalian cell lines. In contrast, thymidine (dThd), either alone or in combination with rNuc-as in EmbryoMax Nucleosides-significantly increases dTTP and dGTP levels, leading to accelerated replication fork progression. Notably, dThd, rather than rNuc, drives fork acceleration and counteracts fork slowdown caused by elevated dUTP, consistent with primer extension assays showing that dUTP transiently inhibits Pol ϵ-mediated DNA synthesis at template adenines. These results clarify the distinct roles of nucleosides in nucleotide metabolism, providing a mechanistic basis for how dThd promotes fork progression and preserves genomic stability.

DNA replication relies on precise coordination between proteins, including the sliding clamp proliferating cell nuclear antigen (PCNA), which encircles DNA to interact with key players in replication and repair. While biochemical studies have demonstrated interactions between PCNA and DNA polymerases δ and ε during DNA synthesis, the functional role of the Pol ε–PCNA interaction in vivo, particularly during leading strand synthesis, remains to be elucidated. To address this question, we employed AlphaFold to model how PCNA interact with four-subunit yeast Pol ε. Our models revealed two distinct points of interaction between Pol ε and PCNA: one at the P-domain and another at a PIP-box, a classical PCNA interaction motif. To validate these findings, we generated mutants that disrupted the Pol ε–PCNA interaction interface. Biochemical assays demonstrated that the PIP-box is critical for this interaction, with the P-domain serving as a secondary contact point. Notably, introducing these mutants into yeast, caused no phenotype in a wild-type background. However, when fewer origins are firing, resulting in longer stretches of leading strand synthesis before forks converge, strains expressing a Pol ε mutant lacking interaction with PCNA showed slower growth. These findings suggest that PCNA enhances the processivity of Pol ε both in vitro and in vivo.

Mitochondrial DNA (mtDNA) stability, essential for cellular energy production, relies on DNA polymerase gamma (POLγ). Here, we show that the POLγ Y951N disease-causing mutation induces replication stalling and severe mtDNA depletion. However, unlike other POLγ disease-causing mutations, Y951N does not directly impair exonuclease activity and only mildly affects polymerase activity. Instead, we found that Y951N compromises the enzyme’s ability to efficiently toggle between DNA synthesis and degradation, and is thus a patient-derived mutation with impaired polymerase-exonuclease switching. These findings provide insights into the intramolecular switch when POLγ proofreads the newly synthesized DNA strand and reveal a new mechanism for causing mitochondrial DNA instability.

The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

Two recent studies provide structural insights into how human DNA polymerase ε (Pol ε) interacts with PCNA to form a processive holoenzyme on the leading strand. A series of cryo-EM images offer structural information on the proofreading process, showing how DNA is transferred between the polymerase and exonuclease sites in human Pol ε.

The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases when building DNA from dNTPs. It is currently believed that DNA polymerases discriminate against NTPs through a steric gate model involving a clash between a tyrosine and the 2′-hydroxyl of the ribonucleotide in the polymerase active site in B-family DNA polymerases. With the help of crystal structures of a B-family polymerase with a UTP or CTP in the active site, molecular dynamics simulations, biochemical assays and yeast genetics, we have identified a mechanism by which the finger domain of the polymerase sense NTPs in the polymerase active site. In contrast to the previously proposed polar filter, our experiments suggest that the amino acid residue in the finger domain senses ribonucleotides by steric hindrance. Furthermore, our results demonstrate that the steric gate in the palm domain and the sensor in the finger domain are both important when discriminating NTPs. Structural comparisons reveal that the sensor residue is conserved among B-family polymerases and we hypothesize that a sensor in the finger domain should be considered in all types of DNA polymerases.

Amino acid substitutions in the exonuclease domain of DNA polymerase (Pol) cause ultramutated tumors. Studies in model organisms suggested pathogenic mechanisms distinct from a simple loss of exonuclease. These mechanisms remain unclear for most recurrent Pol mutations. Particularly, the highly prevalent V411L variant remained a long-standing puzzle with no detectable mutator effect in yeast despite the unequivocal association with ultramutation in cancers. Using purified four-subunit yeast Pol, we assessed the consequences of substitutions mimicking human V411L, S459F, F367S, L424V and D275V. While the effects on exonuclease activity vary widely, all common cancer-Associated variants have increased DNA polymerase activity. Notably, the analog of Pol-V411L is among the strongest polymerases, and structural analysis suggests defective polymerase-To-exonuclease site switching. We further show that the V411L analog produces a robust mutator phenotype in strains that lack mismatch repair, indicating a high rate of replication errors. Lastly, unlike wild-Type and exonuclease-dead Pol, hyperactive variants efficiently synthesize DNA at low dNTP concentrations. We propose that this characteristic could promote cancer cell survival and preferential participation of mutator polymerases in replication during metabolic stress. Our results support the notion that polymerase fitness, rather than low fidelity alone, is an important determinant of variant pathogenicity.

The in vitro reconstitution of origin firing was a key step toward the biochemical reconstitution of eukaryotic DNA replication in budding yeast. Today the basic replication assay involves proteins purified from 24 separate protocols that have evolved since their first publication, and as a result, the efficiency and reliability of the in vitro replication system has improved. Here we will present protocols for all 24 purifications together with a general protocol for the in vitro replication assay and some tips for troubleshooting problems with the assay.

Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase ε (Pol ε) from Saccharomyces cerevisiae, was recently reported to have a [4Fe4S] cluster located within the catalytic domain of the largest subunit, Pol2. Here the redox characteristics of the [4Fe4S] cluster in the context of that domain, Pol2_CORE, are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2_COREexo^–, was used to limit DNA degradation. While no redox signal is apparent for Pol2_COREexo^– on DNA-modified electrodes, a large cathodic signal centered at −140 mV vs NHE is observed after bulk oxidation. A double cysteine to serine mutant (C665S/C668S) of Pol2_COREexo^–, which lacks the [4Fe4S] cluster, shows no similar redox signal upon oxidation. Significantly, protein oxidation yields a sharp decrease in polymerization, while rereduction restores activity almost to the level of untreated enzyme. Moreover, the addition of reduced EndoIII, a bacterial DNA repair enzyme containing [4Fe4S]²⁺, to oxidized Pol2_COREexo^– bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIII^Y82A, a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2_COREexo^–. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2_COREexo^– via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol ε.

Single-stranded DNA breaks, or nicks, are amongst the most common forms of DNA damage in cells. They can be repaired by ligation; however, if a nick occurs just ahead of an approaching replisome, the outcome is a collapsed replication fork comprising a single-ended double-strand break and a 'hybrid nick' with parental DNA on one side and nascent DNA on the other (Figure 1A). We realized that in eukaryotic cells, where replication initiates from multiple replication origins, a fork from an adjacent origin can promote localized re-replication if the hybrid nick is ligated. We have modelled this situation with purified proteins in vitro and have found that there is, indeed, an additional hazard that eukaryotic replisomes face. We discuss how this problem might be mitigated.