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Recent cryo-EM structures of human DNA polymerase ε (Pol ε) bound to PCNA position a Pol ε-specific thumb insertion (polymerase thumb insertion; PTI) adjacent to a PCNA protomer, suggesting a regulatory role in DNA synthesis. To define the functional contribution of this region, we generated alanine-substitution variants in the yeast Pol ε thumb domain, targeting the PTI (SLED1131–1134→AAAA; polε-SLED) and an adjacent conserved loop (PVTE1101–1104→AAAA; polε-PVTE and KPFN1096–1099→AAAA; polε-KPFN). polε-SLED displayed increased intrinsic processivity, efficient bypass of DNA secondary structures, and enhanced synthesis on long templates, consistent with reduced pausing. In contrast, a previous study extended this substitution to six amino acids, SLEDLD1131-1136→AAAAAA, and found a reversed effect, a reduced processivity, indicating that subtle perturbations in this insertion can have opposing functional consequences. polε-PVTE shifted polymerase activity toward exonuclease proofreading and was not fully rescued by PCNA on long templates, whereas polε-KPFN retained near–wild-type activity but showed increased sensitivity to secondary structures that was alleviated by PCNA. In vivo, the corresponding pol2-SLED allele caused a modest mutator phenotype, while pol2-PVTE and pol2-KPFN showed little or no increase. Together, these results indicate that the PTI fine-tunes intrinsic processivity and proofreading to maintain replication fidelity during leading-strand synthesis.

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.

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.

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.

DNA polymerase ϵ (Pol ϵ), the major leading-strand DNA polymerase in eukaryotes, has a catalytic subunit (Pol2) and three non-catalytic subunits. The N-terminal half of Pol2 (Pol2_CORE) exhibits both polymerase and exonuclease activity. It has been suggested that both the non-catalytic C-terminal domain of Pol2 (with the two cysteine motifs CysA and CysB) and Pol2_CORE (with the CysX cysteine motif) are likely to coordinate an Fe–S cluster. Here, we present two new crystal structures of Pol2_CORE with an Fe–S cluster bound to the CysX motif, supported by an anomalous signal at that position. Furthermore we show that purified four-subunit Pol ϵ, Pol ϵ CysA_MUT (C2111S/C2133S), and Pol ϵ CysB_MUT (C2167S/C2181S) all have an Fe–S cluster that is not present in Pol ϵ CysX_MUT (C665S/C668S). Pol ϵ CysA_MUT and Pol ϵ CysB_MUT behave similarly to wild-type Pol ϵ in in vitro assays, but Pol ϵ CysX_MUT has severely compromised DNA polymerase activity that is not the result of an excessive exonuclease activity. Tetrad analyses show that haploid yeast strains carrying CysX_MUT are inviable. In conclusion, Pol ϵ has a single Fe–S cluster bound at the base of the P-domain, and this Fe–S cluster is essential for cell viability and polymerase activity.

The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.

The balance between exonuclease and polymerase activities promotes DNA synthesis over degradation when nucleotides are correctly added to the new strand by replicative B-family polymerases. Misincorporations shift the balance toward the exonuclease site, and the balance tips back in favor of DNA synthesis when the incorrect nucleotides have been removed. Most B-family DNA polymerases have an extended β-hairpin loop that appears to be important for switching from the exonuclease site to the polymerase site, a process that affects fidelity of the DNA polymerase. Here, we show that DNA polymerase ε can switch between the polymerase site and exonuclease site in a processive manner despite the absence of an extended β-hairpin loop. K967 and R988 are two conserved amino acids in the palm and thumb domain that interact with bases on the primer strand in the minor groove at positions n−2 and n−4/n−5, respectively. DNA polymerase ε depends on both K967 and R988 to stabilize the 3′-terminus of the DNA within the polymerase site and on R988 to processively switch between the exonuclease and polymerase sites. Based on a structural alignment with DNA polymerase δ, we propose that arginines corresponding to R988 might have a similar function in other B-family polymerases.

Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3' single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone.