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 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 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 Physical and Functional Interactions of Human DNA Polymerase η with PCNA

2001 ◽  
Vol 21 (21) ◽  
pp. 7199-7206 ◽  
Author(s):  
Lajos Haracska ◽  
Robert E. Johnson ◽  
Ildiko Unk ◽  
Barbara Phillips ◽  
Jerard Hurwitz ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Protein A ◽  
Dna Lesions ◽  
Nuclear Antigen ◽  
Synthetic Activity ◽  
Binding Motif ◽  
Variant Form ◽  
Physical Interaction ◽  
Human Dna ◽  
Factor C

ABSTRACT Human DNA polymerase η (hPolη) functions in the error-free replication of UV-damaged DNA, and mutations in hPolη cause cancer-prone syndrome, the variant form of xeroderma pigmentosum. However, in spite of its key role in promoting replication through a variety of distorting DNA lesions, the manner by which hPolη is targeted to the replication machinery stalled at a lesion site remains unknown. Here, we provide evidence for the physical interaction of hPolη with proliferating cell nuclear antigen (PCNA) and show that mutations in the PCNA binding motif of hPolη inactivate this interaction. PCNA, together with replication factor C and replication protein A, stimulates the DNA synthetic activity of hPolη, and steady-state kinetic studies indicate that this stimulation accrues from an increase in the efficiency of nucleotide insertion resulting from a reduction in the apparentK m for the incoming nucleotide.

Thymine Dimerization Induced by Oxidative DNA Lesions and Epigenetic Intermediates via Triplet-Triplet Energy Transfer

2020 ◽  
Author(s):  
Mauricio Lineros-Rosa ◽  
Antonio Francés-Monerris ◽  
Antonio Monari ◽  
Miguel Angél Miranda ◽  
Virginie Lhiaubet-Vallet
Keyword(s):  
Energy Transfer ◽  
Excitation Energy ◽  
Transient Absorption ◽  
Theoretical Calculations ◽  
Dna Lesions ◽  
Transient Absorption Spectroscopy ◽  
Triplet Energy ◽  
Pyrimidine Dimers ◽  
Triplet Energy Transfer ◽  
Dna Nucleobases

Interaction of nucleic acids with light is a scientific question of paramount relevance not only in the understanding of life functioning and evolution, but also in the insurgence of diseases such as malignant skin cancer and in the development of biomarkers and novel light-assisted therapeutic tools. This work shows that the UVA portion of sunlight, not absorbed by canonical DNA nucleobases, can be absorbed by 5-formyluracil (ForU) and 5-formylcytosine (ForC), two ubiquitous oxidative lesions and epigenetic intermediates present in living beings in natural conditions. We measure the strong propensity of these molecules to populate triplet excited states able to transfer the excitation energy to thymine-thymine dyads, inducing the formation of the highly toxic and mutagenic cyclobutane pyrimidine dimers (CPDs). By using steady-state and transient absorption spectroscopy, NMR, HPLC, and theoretical calculations, we quantify the differences in the triplet-triplet energy transfer mediated by ForU and ForC, revealing that the former is much more efficient in delivering the excitation energy and producing the CPD photoproduct. Although significantly slower than ForU, ForC is also able to harm DNA nucleobases and therefore this process has to be taken into account as a viable photosensitization mechanism. The present findings evidence a rich photochemistry crucial to understand DNA photodamage and of potential use in the development of biomarkers and non-conventional photodynamic therapy agents.

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.

The role of p53 in regulating genomic stability when DNA and RNA synthesis are inhibited

1995 ◽  
Vol 20 (10) ◽  
pp. 431-434 ◽  
Author(s):  
Olga B. Chernova ◽  
Michail V. Chernov ◽  
Munna L. Agarwal ◽  
William R. Taylor ◽  
George R. Stark
Keyword(s):  
Rna Synthesis ◽  
Genomic Stability ◽  
Dna And Rna ◽  
Dna And Rna Synthesis

Benzamide on chondrocytic differentiation in chick limb bud cell culture

Development ◽  
1985 ◽  
Vol 85 (1) ◽  
pp. 163-175
Author(s):  
Shinobu Nakanishi ◽  
Edwin M. Uyeki
Keyword(s):  
Cell Culture ◽  
Cell Proliferation ◽  
Trichloroacetic Acid ◽  
Rna Synthesis ◽  
Limb Bud ◽  
The Other ◽  
Transferase Activity ◽  
Dna And Rna ◽  
Atp Levels ◽  
Chondrocytic Differentiation

Benzamide, an inhibitor of (ADP-ribose) transferase, augmented chondrocytic differentiation of chick limb bud mesenchymal cells in micromass cultures; the incorporation of 35SO42− into the trichloroacetic-acid-insoluble constituents of cell masses as well as the formation of cartilage nodules (Nishio, Nakanishi, Doull & Uyeki, 1983) occurred about 24h earlier than in untreated cultures and continued to be enhanced in benzamide-treated cultures of stage 23- to 24-chick limb bud cells. Benzamide also significantly increased cell proliferation. However, benzamide did not affect DNA and RNA syntheses except for one period: 24 to 30 h after the start of culture, RNA synthesis was stimulated. From 48h of culture, (ADP-ribose) transferase activity decreased daily in untreated cultures, whereas benzamide treatment diminished (ADP-ribose) transferase activity 24 h earlier. On the other hand, intracellular NAD levels increased daily in untreated cultures, and benzamide significantly increased the NAD levels above untreated cultures. ATP levels did not differ significantly during the culture period, and benzamide did not affect ATP levels.

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