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.
Abstract
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.
Graphical Abstract
Graphical Abstract
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 (Pol2CORE) 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 Pol2CORE (with the CysX cysteine motif) are likely to coordinate an Fe-S cluster. Here, we present two new crystal structures of Pol2CORE 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 CysAMUT (C2111S/C2133S), and Pol ϵ CysBMUT (C2167S/C2181S) all have an Fe-S cluster that is not present in Pol ϵ CysXMUT (C665S/C668S). Pol ϵ CysAMUT and Pol ϵ CysBMUT behave similarly to wild-type Pol ϵ in in vitro assays, but Pol ϵ CysXMUT has severely compromised DNA polymerase activity that is not the result of an excessive exonuclease activity. Tetrad analyses show that haploid yeast strains carrying CysXMUT 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 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.
DNA polymerase ɛ (Pol ɛ) is a high-fidelity polymerase that has been shown to participate in leading-strand synthesis during DNA replication in eukaryotic cells. We present here a ternary structure ...of the catalytic core of Pol ɛ (142 kDa) from Saccharomyces cerevisiae in complex with DNA and an incoming nucleotide. This structure provides information about the selection of the correct nucleotide and the positions of amino acids that might be critical for proofreading activity. Pol ɛ has the highest fidelity among B-family polymerases despite the absence of an extended β-hairpin loop that is required for high-fidelity replication by other B-family polymerases. Moreover, the catalytic core has a new domain that allows Pol ɛ to encircle the nascent double-stranded DNA. Altogether, the structure provides an explanation for the high processivity and high fidelity of leading-strand DNA synthesis in eukaryotes.
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.
•A primary 939±3Ma remanent magnetization is here reported from SE Sweden.•A new 945–935Ma key pole for Baltica is presented.•Implications for the Sveconorwegian and Grenvillian loops are ...discussed.•Slow low latitudinal drift for Baltica and Laurentia between 960 and 920Ma.
We present the results of palaeomagnetic and 40Ar/39Ar studies of the Proterozoic mafic dykes in the Norrköping and Falun areas of the southern Sweden. The primary remanence of two 939±3Ma dykes is supported by the rigorous baked contact test. The remanence direction of two other dykes, one of which was previously U–Pb dated at 946±1Ma is close to the reverse direction of 939Ma dykes. Using these results together with previously published 935±5Ma palaeomagnetic data from the Göteborg-Slussen mafic dykes and some dykes from the Falun area we calculated the mean 946–935Ma palaeopole for Baltica (0.9°S, 240.7°E, A95=6.7), which can be qualified as the key pole. Using this pole together with other date we conclude that the Grenville and Sveconorwegian loops of Laurentian and Baltican Apparent Polar Wander Paths are temporary displaced by 100–150m.y. We propose new palaeogeographic reconstructions of Baltica and Laurentia at ca. 940Ma and ca. 850Ma. We also present two new Mesoproterozoic non-key poles from 1410Ma and 1595Ma dykes.
PDF
