Proofreading exonuclease activity of human DNA polymerase delta and its effects on lesion-bypass DNA synthesis.
Ontology highlight
ABSTRACT:
SUBMITTER: Fazlieva R
PROVIDER: S-EPMC2685094 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Replicative DNA polymerases (DNA pols) increase their fidelity by removing misincorporated nucleotides with their 3' ? 5' exonuclease activity. Exonuclease activity reduces translesion synthesis (TLS) efficiency and TLS DNA pols lack 3' ? 5' exonuclease activity. Here we show that physiological concentrations of pyrophosphate (PP(i)) activate the pyrophosphorolytic activity by DNA pol-?, allowing the preferential excision of the incorrectly incorporated A opposite a 7,8-dihydro-8-oxoguanine lesion, or T opposite a 6-methyl-guanine, with respect to the correct C. This is the first example of an alternative proofreading mechanism used during TLS.
| S-EPMC3246252 | biostudies-literature
Project description:Base selectivity, proofreading, and postreplication mismatch repair are important for replication fidelity. Because proofreading plays an important role in error correction, we have investigated factors that influence its impact in the yeast Saccharomyces cerevisiae. We have utilized a sensitive mutation detection system based on homonucleotide runs of 4 to 14 bases to examine the impact of DNA polymerase delta proofreading on mutation avoidance. The contribution of DNA polymerase delta proofreading on error avoidance was found to be similar to that of DNA polymerase epsilon proofreading in short homonucleotide runs (A4 and A5) but much greater than the contribution of DNA polymerase epsilon proofreading in longer runs. We have identified an intraprotein interaction affecting mutation prevention that results from mutations in the replication and the proofreading regions, resulting in an antimutator phenotype relative to a proofreading defect. Finally, a diploid strain with a defect in DNA polymerase delta proofreading exhibits a higher mutation rate than a haploid strain. We suggest that in the diploid population of proofreading defective cells there exists a transiently hypermutable fraction that would be inviable if cells were haploids.
| S-EPMC1460598 | biostudies-other
Project description:Prior sequence analysis studies have suggested that bacterial ribonuclease (RNase) Ds comprise a complete domain that is found also in Homo sapiens polymyositis-scleroderma overlap syndrome 100 kDa autoantigen and Werner syndrome protein. This RNase D 3'-->5' exoribonuclease domain was predicted to have a structure and mechanism of action similar to the 3'-->5' exodeoxyibonuclease (proofreading) domain of DNA polymerases. Here, hidden Markov model (HMM) and phylogenetic studies have been used to identify and characterise other sequences that may possess this exonuclease domain. Results indicate that it is also present in the RNase T family; Borrelia burgdorferi P93 protein, an immunodominant antigen in Lyme disease; bacteriophage T4 dexA and Escherichia coli exonuclease I, processive 3'-->5' exodeoxyribonucleases that degrade single-stranded DNA; Bacillus subtilis dinG, a probable helicase involved in DNA repair and possibly replication, and peptide synthase 1; Saccharomyces cerevisiae Pab1p-dependent poly(A) nuclease PAN2 subunit, required for shortening mRNA poly(A) tails; Caenorhabditis elegans and Mus musculus CAF1, transcription factor CCR4-associated factor 1; Xenopus laevis XPMC2, prevention of mitotic catastrophe in fission yeast; Drosophila melanogaster egalitarian, oocyte specification and axis determination, and exuperantia, establishment of oocyte polarity; H.sapiens HEM45, expressed in tumour cell lines and uterus and regulated by oestrogen; and 31 open reading frames including one in Methanococcus jannaschii . Examination of a multiple sequence alignment and two three-dimensional structures of proofreading domains has allowed definition of the core sequence, structural and functional elements of this exonuclease domain.
| S-EPMC147149 | biostudies-other
Project description:Nucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase iota was measured and structures of the DNA polymerase iota-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase iota opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase iota incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase iota opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase iota extends from the N2-ethyl-Gua:Cyt 3' terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase iota catalyzed reaction is well suited for N(2)-ethyl-Gua bypass. The structure of DNA polymerase iota with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase iota complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase iota polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase iota efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.
| S-EPMC2615510 | biostudies-literature
Project description:Mutations are a hallmark of cancer. Normal cells minimize spontaneous mutations through the combined actions of polymerase base selectivity, 3' --> 5' exonucleolytic proofreading, mismatch correction, and DNA damage repair. To determine the consequences of defective proofreading in mammals, we created mice with a point mutation (D400A) in the proofreading domain of DNA polymerase delta (poldelta, encoded by the Pold1 gene). We show that this mutation inactivates the 3' --> 5' exonuclease of poldelta and causes a mutator and cancer phenotype in a recessive manner. By 18 months of age, 94% of homozygous Pold1(D400A/D400A) mice developed cancer and died (median survival = 10 months). In contrast, only 3-4% of Pold1(+/D400A) and Pold1(+/+) mice developed cancer in this time frame. Of the 66 tumors arising in 49 Pold1(D400A/D400A) mice, 40 were epithelial in origin (carcinomas), 24 were mesenchymal (lymphomas and sarcomas), and two were composite (teratomas); one-third of these animals developed tumors in more than one tissue. Skin squamous cell carcinoma was the most common tumor type, occurring in 60% of all Pold1(D400A/D400A) mice and in 90% of those surviving beyond 8 months of age. These data show that poldelta proofreading suppresses spontaneous tumor development and strongly suggest that unrepaired DNA polymerase errors contribute to carcinogenesis. Mice deficient in poldelta proofreading provide a tractable model to study mechanisms of epithelial tumorigenesis initiated by a mutator phenotype.
| S-EPMC137756 | biostudies-literature
Project description:A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant alkylative DNA lesion. If not repaired, Fm7dG can undergo spontaneous depurination, imidazole ring-opening, or bypass by translesion synthesis DNA polymerases. Human DNA polymerase η (polη) efficiently catalyzes across Fm7dG in vitro, but its structural basis is unknown. Herein, we report a crystal structure of polη in complex with templating Fm7dG and an incoming nonhydrolyzable dCTP analog, where a 2'-fluorine-mediated transition destabilization approach was used to prevent the spontaneous depurination of Fm7dG. The structure showed that polη readily accommodated the Fm7dG:dCTP base pair with little conformational change of protein and DNA. In the catalytic site, Fm7dG and dCTP formed three hydrogen bonds with a Watson-Crick geometry, indicating that the major keto tautomer of Fm7dG is involved in base pairing. The polη-Fm7dG:dCTP structure was essentially identical to the corresponding undamaged structure, which explained the efficient bypass of the major methylated lesion. Overall, the first structure of translesion synthesis DNA polymerase bypassing Fm7dG suggests that in the catalytic site of Y-family DNA polymerases, small N7-alkylguanine adducts may be well tolerated and form the canonical Watson-Crick base pair with dCTP through their keto tautomers.
| S-EPMC6864850 | biostudies-literature
Project description:Leading-strand DNA replication by polymerase epsilon (Polϵ) across single-strand breaks (SSBs) causes single-ended double-strand breaks (seDSBs), which are repaired via homology-directed repair (HDR) and suppressed by fork reversal (FR). Although previous studies identified many molecules required for hydroxyurea-induced FR, FR at seDSBs is poorly understood. Here, we identified molecules that specifically mediate FR at seDSBs. Because FR at seDSBs requires poly(ADP ribose)polymerase 1 (PARP1), we hypothesized that seDSB/FR-associated molecules would increase tolerance to camptothecin (CPT) but not the PARP inhibitor olaparib, even though both anti-cancer agents generate seDSBs. Indeed, we uncovered that Polϵ exonuclease and CTF18, a Polϵ cofactor, increased tolerance to CPT but not olaparib. To explore potential functional interactions between Polϵ exonuclease, CTF18, and PARP1, we created exonuclease-deficient POLE1exo-/-, CTF18-/-, PARP1-/-, CTF18-/-/POLE1exo-/-, PARP1-/-/POLE1exo-/-, and CTF18-/-/PARP1-/- cells. Epistasis analysis indicated that Polϵ exonuclease and CTF18 were interdependent and required PARP1 for CPT tolerance. Remarkably, POLE1exo-/- and HDR-deficient BRCA1-/- cells exhibited similar CPT sensitivity. Moreover, combining POLE1exo-/- with BRCA1-/- mutations synergistically increased CPT sensitivity. In conclusion, the newly identified PARP1-CTF18-Polϵ exonuclease axis and HDR act independently to prevent fork collapse at seDSBs. Olaparib inhibits this axis, explaining the pronounced cytotoxic effects of olaparib on HDR-deficient cells.
| S-EPMC10711444 | biostudies-literature
Project description:Sulfolobus solfataricus DNA Polymerase IV (Dpo4), a prototype Y-family DNA polymerase, has been well characterized biochemically and biophysically at 37 °C or lower temperatures. However, the physiological temperature of the hyperthermophile S. solfataricus is approximately 80 °C. With such a large discrepancy in temperature, the in vivo relevance of these in vitro studies of Dpo4 has been questioned. Here, we employed circular dichroism spectroscopy and fluorescence-based thermal scanning to investigate the secondary structural changes of Dpo4 over a temperature range from 26 to 119 °C. Dpo4 was shown to display a high melting temperature characteristic of hyperthermophiles. Unexpectedly, the Little Finger domain of Dpo4, which is only found in the Y-family DNA polymerases, was shown to be more thermostable than the polymerase core. More interestingly, Dpo4 exhibited a three-state cooperative unfolding profile with an unfolding intermediate. The linker region between the Little Finger and Thumb domains of Dpo4 was found to be a source of structural instability. Through site-directed mutagenesis, the interactions between the residues in the linker region and the Palm domain were identified to play a critical role in the formation of the unfolding intermediate. Notably, the secondary structure of Dpo4 was not altered when the temperature was increased from 26 to 87.5 °C. Thus, in addition to providing structural insights into the thermal stability and an unfolding intermediate of Dpo4, our work also validated the relevance of the in vitro studies of Dpo4 performed at temperatures significantly lower than 80 °C.
| S-EPMC3398204 | biostudies-literature
Project description:Organisms require faithful DNA replication to avoid deleterious mutations. In yeast, replicative leading- and lagging-strand DNA polymerases (Pols epsilon and delta, respectively) have intrinsic proofreading exonucleases that cooperate with each other and mismatch repair to limit spontaneous mutation to less than 1 per genome per cell division. The relationship of these pathways in mammals and their functions in vivo are unknown. Here we show that mouse Pol epsilon and delta proofreading suppress discrete mutator and cancer phenotypes. We found that inactivation of Pol epsilon proofreading elevates base-substitution mutations and accelerates a unique spectrum of spontaneous cancers; the types of tumors are entirely different from those triggered by loss of Pol delta proofreading. Intercrosses of Pol epsilon-, Pol delta-, and mismatch repair-mutant mice show that Pol epsilon and delta proofreading act in parallel pathways to prevent spontaneous mutation and cancer. These findings distinguish Pol epsilon and delta functions in vivo and reveal tissue-specific requirements for DNA replication fidelity.
| S-EPMC2761330 | biostudies-literature
Project description:The Y-family DNA polymerase REV1 is involved in replicative bypass of damaged DNA and G-quadruplex (G4) DNA. In addition to a scaffolding role in the replicative bypass, REV1 acts in a catalytic role as a deoxycytidyl transferase opposite some replication stall sites, e.g., apurinic/apyrimidinic (AP) sites, N(2)-guanyl lesions, and G4 sites. We characterized the biochemical properties of 12 reported germline missense variants of human REV1, including the N373S variant associated with high risk of cervical cancer, using the recombinant REV1 (residues 330-833) proteins and DNA templates containing a G, AP site, N(2)-CH2(2-naphthyl)G (N(2)-NaphG), or G4. In steady-state kinetic analyses, the F427L, R434Q, M656V, D700N, R704Q, and P831L variants displayed 2- to 8-fold decreases in kcat/Km for dCTP insertion opposite all four templates, compared to that of wild-type, while the N373S, M407L, and N497S showed 2- to 3-fold increases with all four and the former three or two templates, respectively. The F427L, R434Q, M656V, and R704Q variants also had 2- to 3-fold lower binding affinities to DNA substrates containing G, an AP site, and/or N(2)-NaphG than wild-type. Distinctively, the N373S variant had a 3-fold higher binding affinity to G4 DNA than the wild-type, as well as a 2-fold higher catalytic activity opposite the first tetrad G, suggesting a facilitating effect of this variation on replication of G4 DNA sequences in certain human papillomavirus genomes. Our results suggest that the catalytic function of REV1 is moderately or slightly altered by at least nine genetic variations, and the G4 DNA processing function of REV1 is slightly enhanced by the N373S variation, which might provide the possibility that certain germline missense REV1 variations affect the individual susceptibility to carcinogenesis by modifying the capability of REV1 for replicative bypass past DNA lesions and G4 motifs derived from chemical and viral carcinogens.
| S-EPMC5116390 | biostudies-literature