scholarly journals Selective Metal Ion Utilization Contributes to the Transformation of the Activity of Yeast Polymerase η from DNA Polymerization toward RNA Polymerization

2020 ◽  
Vol 21 (21) ◽  
pp. 8248
Author(s):  
Eva Balint ◽  
Ildiko Unk
Keyword(s):  
Dna Synthesis ◽  
Metal Ion ◽  
Rna Synthesis ◽  
Dna Lesions ◽  
Magnesium Concentration ◽  
Synthetic Activity ◽  
Polymerase Eta ◽  
Rna Polymerization ◽  
Metal Cofactor ◽  
Fold Excess

Polymerase eta (Polη) is a translesion synthesis DNA polymerase directly linked to cancer development. It can bypass several DNA lesions thereby rescuing DNA damage-stalled replication complexes. We previously presented evidence implicating Saccharomyces cerevisiae Polη in transcription elongation, and identified its specific RNA extension and translesion RNA synthetic activities. However, RNA synthesis by Polη proved rather inefficient under conditions optimal for DNA synthesis. Searching for factors that could enhance its RNA synthetic activity, we have identified the divalent cation of manganese. Here, we show that manganese triggers drastic changes in the activity of Polη. Kinetics experiments indicate that manganese increases the efficiency of ribonucleoside incorporation into RNA by ~400–2000-fold opposite undamaged DNA, and ~3000 and ~6000-fold opposite TT dimer and 8oxoG, respectively. Importantly, preference for the correct base is maintained with manganese during RNA synthesis. In contrast, activity is strongly impaired, and base discrimination is almost lost during DNA synthesis by Polη with manganese. Moreover, Polη shows strong preference for manganese during RNA synthesis even at a 25-fold excess magnesium concentration. Based on this, we suggest that a new regulatory mechanism, selective metal cofactor utilization, modulates the specificity of Polη helping it to perform distinct activities needed for its separate functions during replication and transcription.

scholarly journals Selective metal ion utilization contributes to the transformation of the activity of yeast polymerase η from DNA polymerization toward RNA polymerization

2020 ◽  
Author(s):  
Eva Balint ◽  
Ildiko Unk
Keyword(s):  
Dna Synthesis ◽  
Metal Ion ◽  
Rna Synthesis ◽  
Dna Lesions ◽  
Magnesium Concentration ◽  
Synthetic Activity ◽  
Polymerase Eta ◽  
Rna Polymerization ◽  
Metal Cofactor ◽  
Fold Excess

ABSTRACTPolymerase eta (Polη) is a translesion synthesis DNA polymerase directly linked to cancer development. It can bypass several DNA lesions thereby rescuing DNA damage-stalled replication complexes. We previously presented evidence implicating Saccharomyces cerevisiae Polη in transcription elongation, and identified its specific RNA extension and translesion RNA synthetic activities. However, RNA synthesis by Polη proved rather inefficient under conditions optimal for DNA synthesis. Searching for factors that could enhance its RNA synthetic activity, we have identified the divalent cation of manganese. Here we show, that manganese triggers drastic changes in the activity of Polη. It increases the efficiency of ribonucleoside incorporation into RNA by ∼400-2000-fold opposite undamaged DNA, and ∼3000 and ∼6000-fold opposite TT dimer and 8oxoG, respectively. Importantly, preference for the correct base is maintained with manganese during RNA synthesis. In contrast, activity is strongly impaired, and base discrimination almost lost during DNA synthesis by Polη with manganese. Moreover, Polη shows strong preference for manganese during RNA synthesis even at 25-fold excess magnesium concentration. Based on these, we suggest that selective metal cofactor usage plays an important role in determining the specificity of Polη during synthesis enabling it to function at both replication and transcription.

scholarly journals Manganese Is a Strong Specific Activator of the RNA Synthetic Activity of Human Polη

2021 ◽  
Vol 23 (1) ◽  
pp. 230
Author(s):  
Eva Balint ◽  
Ildiko Unk
Keyword(s):  
Rna Synthesis ◽  
Dna Lesions ◽  
Chain Extension ◽  
Synthetic Activity ◽  
Variant Form ◽  
Lower Efficiency ◽  
Dna And Rna ◽  
Pyrimidine Dimers ◽  
Metal Cofactor ◽  
Order Of Magnitude

DNA polymerase η (Polη) is a translesion synthesis polymerase that can bypass different DNA lesions with varying efficiency and fidelity. Its most well-known function is the error-free bypass of ultraviolet light-induced cyclobutane pyrimidine dimers. The lack of this unique ability in humans leads to the development of a cancer-predisposing disease, the variant form of xeroderma pigmentosum. Human Polη can insert rNTPs during DNA synthesis, though with much lower efficiency than dNTPs, and it can even extend an RNA chain with ribonucleotides. We have previously shown that Mn2+ is a specific activator of the RNA synthetic activity of yeast Polη that increases the efficiency of the reaction by several thousand-fold over Mg2+. In this study, our goal was to investigate the metal cofactor dependence of RNA synthesis by human Polη. We found that out of the investigated metal cations, only Mn2+ supported robust RNA synthesis. Steady state kinetic analysis showed that Mn2+ activated the reaction a thousand-fold compared to Mg2+, even during DNA damage bypass opposite 8-oxoG and TT dimer. Our results revealed a two order of magnitude higher affinity of human Polη towards ribonucleotides in the presence of Mn2+ compared to Mg2+. It is noteworthy that activation occurred without lowering the base selectivity of the enzyme on undamaged templates, whereas the fidelity decreased across a TT dimer. In summary, our data strongly suggest that, like with its yeast homolog, Mn2+ is the proper metal cofactor of hPolη during RNA chain extension, and selective metal cofactor utilization contributes to switching between its DNA and RNA synthetic activities.

scholarly journals Non-Conventional Metal Ion Cofactor Requirement of Dinoflagellate Alkaline Phosphatase and Translational Regulation by Phosphorus Limitation

2019 ◽  
Vol 7 (8) ◽  
pp. 232 ◽  
Author(s):  
Xin Lin ◽  
Chentao Guo ◽  
Ling Li ◽  
Tangcheng Li ◽  
Senjie Lin
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Ion ◽  
Translational Regulation ◽  
Organic Phosphorus ◽  
Niche Differentiation ◽  
Phosphorus Limitation ◽  
Marine Phytoplankton ◽  
Phosphate Limitation ◽  
Dissolved Organic Phosphorus ◽  
Metal Cofactor

Alkaline phosphatase (AP) enables marine phytoplankton to utilize dissolved organic phosphorus (DOP) when dissolved inorganic phosphate (DIP) is depleted in the ocean. Dinoflagellate AP (Dino-AP) represents a newly classified atypical type of AP, PhoAaty. Despite While being a conventional AP, PhoAEC is known to recruit Zn2+ and Mg2+ in the active center, and the cofactors required by PhoAaty have been contended and remain unclear. In this study, we investigated the metal ion requirement of AP in five dinoflagellate species. After AP activity was eliminated by using EDTA to chelate metal ions, the enzymatic activity could be recovered by the supplementation of Ca2+, Mg2+ and Mn2+ in all cases but not by that of Zn2+. Furthermore, the same analysis conducted on the purified recombinant ACAAP (AP of Amphidinium carterae) verified that the enzyme could be activated by Ca2+, Mg2+, and Mn2+ but not Zn2+. We further developed an antiserum against ACAAP, and a western blot analysis using this antibody showed a remarkable up-regulation of ACAAP under a phosphate limitation, consistent with elevated AP activity. The unconventional metal cofactor requirement of Dino-AP may be an adaptation to trace metal limitations in the ocean, which warrants further research to understand the niche differentiation between dinoflagellates and other phytoplankton that use Zn–Mg AP in utilizing DOP.

scholarly journals RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1631-1640 ◽  
Author(s):  
Janet R Donaldson ◽  
Charmain T Courcelle ◽  
Justin Courcelle
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Dna Lesions ◽  
Replication Forks ◽  
Wild Type ◽  
Replication Machinery ◽  
Dna Junctions ◽  
Fork Regression

Abstract Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

High-resolution autoradiographic localization of DNA-containing sites and RNA synthesis in developing nucleoli of human preimplantation embryos: a new concept of embryonic nucleologenesis

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 777-791 ◽  
Author(s):  
J. Tesarik ◽  
V. Kopecny ◽  
M. Plachot ◽  
J. Mandelbaum
Keyword(s):  
Rna Synthesis ◽  
Preimplantation Embryos ◽  
Synthetic Activity ◽  
Rrna Synthesis ◽  
Nucleolar Organizer Regions ◽  
Careful Study ◽  
Matrix Elements ◽  
Electron Microscopic Autoradiography ◽  
General Validity ◽  
Granular Component

Human embryos from the 2-cell to the morula stage, obtained by in vitro fertilization, were incubated with [3H]thymidine or [3H]uridine so as to achieve labelling of all replicating nuclear DNA and the newly synthesized RNA, respectively. The label was localized in different structural components of developing nucleoli using electron microscopic autoradiography. Careful study of the relationship between the structural pattern and nucleic acid distribution made it possible to define four stages of embryonic nucleologenesis. Homogeneous nuclear precursors (i) consist of nucleolar matrix elements appearing as filaments of 3 nm thickness, (ii) do not contain recently replicated DNA and (iii) lack RNA synthetic activity. Penetration of DNA into these bodies is a key event leading to their transformation into heterogeneous nucleolar precursors. In addition to the 3 nm matrix filaments, two types of 5 nm fibrillar components can be recognized in them. The denser type contains DNA and is the site of nucleolar RNA synthesis, while the more loosely arranged 5 nm fibrils are not labelled with [3H]thymidine and apparently represent the newly produced pre-rRNA detached from the transcribing rDNA filament. Compact fibrillogranular nucleoli are characterized by the first appearance of the granular component and reduction of the nontranscribing part of the fibrillar component, both indicating the activation of the machinery for rRNA processing. Finally, the granular component is most evident in reticulated nucleoli, occupying mostly the inner parts of their nucleolonema, while the transcription sites tend to be located at the nucleolar periphery. Our findings advocate a unique concept of embryonic nucleologenesis, different from any other nucleolar event during the cell cycle of differentiated cells. This developmental pattern is characterized by a gradual activation of rRNA synthesis and processing, mediated by progressive association of rDNA and, later on, the newly formed pre-rRNA with pre-existing nucleolar matrix elements that are originally topically separated from nucleolar organizer regions. This model may have a general validity in early animal embryos despite some interspecies variability in the timing of individual steps and resulting structural peculiarities.

scholarly journals Enzymatic RNA Production from NTPs Synthesized from Nucleosides and Trimetaphosphate

2021 ◽  
Author(s):  
Fabio Chizzolini ◽  
Alexandra Kent ◽  
Luiz F. M. Passalacqua ◽  
Andrej Lupták
Keyword(s):  
Rna Synthesis ◽  
Rna World ◽  
Rna Replication ◽  
High Energy ◽  
Rna Degradation ◽  
T7 Rna Polymerase ◽  
Nucleotide Synthesis ◽  
Enthalpy Of Activation ◽  
Rna Polymerization ◽  
Synthetic Approaches

<p>A mechanism of nucleoside triphosphorylation would have been critical in an evolving “RNA world” to provide high-energy substrates for reactions such as RNA polymerization. However, synthetic approaches to produce ribonucleoside triphosphoates (rNTPs) have suffered from conditions such as high temperatures or high pH that lead to increased RNA degradation, as well as substrate production that cannot sustain replication. We demonstrate that cyclic trimetaphosphate (cTmp) can react with nucleosides to form rNTPs under mild, prebiotically-relevant conditions, with second-order rate constants ranging from 1.7 x 10<sup>–6</sup> to 6.5 x 10<sup>–6</sup> M<sup>–1</sup> s<sup>–1</sup>. The ATP reaction shows a linear dependence on pH and Mg<sup>2+</sup>, and an enthalpy of activation of 88 ± 4 kJ/mol. At millimolar nucleoside and cTmp concentrations, the rNTP production rate is sufficient to facilitate RNA synthesis by both T7 RNA polymerase and a polymerase ribozyme. We suggest that the optimized reaction of cTmp with nucleosides may provide a viable connection between prebiotic nucleotide synthesis and RNA replication.</p>

scholarly journals RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1738
Author(s):  
Alesia A. Levanova ◽  
Eeva J. Vainio ◽  
Jarkko Hantula ◽  
Minna M. Poranen
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Polymerization Reaction ◽  
Rna Dependent Rna Polymerase ◽  
Independent Manner ◽  
Nucleotidyl Transferase ◽  
Rna Polymerization ◽  
The Family

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.

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