| 1. |
- Barbari, Stephanie R., et al.
(författare)
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Enhanced polymerase activity permits efficient synthesis by cancer-Associated DNA polymerase variants at low dNTP levels
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:14, s. 8023-8040
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Jokipii-Lukkari, Soile, et al.
(författare)
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Dual targeted poplar ferredoxin NADP+ oxidoreductase interacts with hemoglobin 1
- 2016
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Ingår i: Plant Science. - : Elsevier BV. - 0168-9452. ; 247, s. 138-149
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Tidskriftsartikel (refereegranskat)abstract
- Previous reports have connected non-symbiotic and truncated hemoglobins (Hbs) to metabolism of nitric oxide (NO), an important signalling molecule involved in wood formation. We have studied the capability of poplar (Populus tremula × tremuloides) Hbs PttHb1 and PttTrHb proteins alone or with a flavin-protein reductase to relieve NO cytotoxicity in living cells. Complementation tests in a Hb-deficient, NO-sensitive yeast (Saccharomyces cerevisiae) δyhb1 mutant showed that neither PttHb1 nor PttTrHb alone protected cells against NO. To study the ability of Hbs to interact with a reductase, ferredoxin NADP+ oxidoreductase PtthFNR was characterized by sequencing and proteomics. To date, by far the greatest number of the known dual-targeted plant proteins are directed to chloroplasts and mitochondria. We discovered a novel variant of hFNR that lacks the plastid presequence and resides in cytosol. The coexpression of PttHb1 and PtthFNR partially restored NO resistance of the yeast δyhb1 mutant, whereas PttTrHb coexpressed with PtthFNR failed to rescue growth. YFP fusion proteins confirmed the interaction between PttHb1 and PtthFNR in plant cells. The structural modelling results indicate that PttHb1 and PtthFNR are able to interact as NO dioxygenase. This is the first report on dual targeting of central plant enzyme FNR to plastids and cytosol.
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| 3. |
- Parkash, Vimal, et al.
(författare)
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A sensor complements the steric gate when DNA polymerase ε discriminates ribonucleotides
- 2023
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 51:20, s. 11225-11238
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Tidskriftsartikel (refereegranskat)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.
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| 4. |
- Parkash, Vimal, et al.
(författare)
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Structural consequence of the most frequently recurring cancer-associated substitution in DNA polymerase epsilon
- 2019
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The most frequently recurring cancer-associated DNA polymerase epsilon (Pol epsilon) mutation is a P286R substitution in the exonuclease domain. While originally proposed to increase genome instability by disrupting exonucleolytic proofreading, the P286R variant was later found to be significantly more pathogenic than Pol epsilon proofreading deficiency per se. The mechanisms underlying its stronger impact remained unclear. Here we report the crystal structure of the yeast orthologue, Pol epsilon-P301R, complexed with DNA and an incoming dNTP. Structural changes in the protein are confined to the exonuclease domain, with R301 pointing towards the exonuclease site. Molecular dynamics simulations suggest that R301 interferes with DNA binding to the exonuclease site, an outcome not observed with the exonuclease-inactive Pol epsilon-D290A, E292A variant lacking the catalytic residues. These results reveal a distinct mechanism of exonuclease inactivation by the P301R substitution and a likely basis for its dramatically higher mutagenic and tumorigenic effects.
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| 5. |
- ter Beek, Josy, et al.
(författare)
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Structural evidence for an essential Fe–S cluster in the catalytic core domain of DNA polymerase ϵ
- 2019
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 47:11, s. 5712-5722
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Tidskriftsartikel (refereegranskat)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.
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