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Endonuclease activities from a permanently established mouse cell line that act upon DNA damaged by ultraviolet
Abstract:
The activity of damage-dependent endonuclease in mouse plasmacytoma cells (line MPC-11) has been studied using damaged phi X 174 RFI DNA as substrate. The DNA was treated with ultraviolet light, acid, or osmium tetroxide to introduce different types of lesions. Ultraviolet light-damaged DNA was cleaved at approx. 1.1 sites per 35 thymine-containing dimers by the extract, which indicates no specificity towards this type of lesion. The acid-treated DNA, which contains apurinic sites, was enzymatically broken in every alkalilabile site and this strongly suggests the presence of an apurinic-specific endonuclease activity in the nuclear extract. The activity which acts on ultraviolet-irradiated DNA and that which acts on acid-treated DNA have different specificities as shown by their salt requirements and the extent to which they are stimulated by magnesium. While the ultraviolet-endonuclease activity was very little affected by reducing the KCl concentration, the apurinic-specific activity was almost completely abolished. Osmium tetroxide renders the DNA an excellent substrate for endonucleolytic activity in the mouse cell extract. The response to KCl and MgCl2 of the osmium tetroxide-specific endonuclease activity is qualitatively similar to that of the endonuclease activity, which acts on ultraviolet-irradiated DNA. Treatment of DNA with osmium tetroxide is known to produce 5,6-dihydroxydihydrothymine which is a minor photoproduct in DNA after irradiation, suggesting that the ultraviolet-specific endonuclease activity acts upon this lesion.
Insights
Mouse plasmacytoma cells possess damage-dependent endonuclease activity. This enzyme shows specificity for apurinic sites in DNA, with distinct salt and magnesium requirements compared to its response to UV-damaged DNA.
Area of Science:
- Molecular Biology
- Biochemistry
- DNA Repair Mechanisms
Background:
- DNA damage can arise from various sources, including UV radiation and chemical treatments.
- Endonucleases play a crucial role in DNA repair pathways by cleaving damaged DNA strands.
- Understanding the specificity of these enzymes is vital for elucidating DNA repair processes.
Purpose of the Study:
- To investigate the damage-dependent endonuclease activity in mouse plasmacytoma cells (MPC-11).
- To characterize the substrate specificity and biochemical properties of the identified endonuclease activities.
- To differentiate between endonucleases acting on UV-induced lesions versus apurinic sites.
Main Methods:
- Utilized damaged phi X 174 RFI DNA as a substrate, introducing lesions via UV light, acid, or osmium tetroxide treatment.
- Assessed endonuclease activity using nuclear extracts from mouse plasmacytoma cells.
- Analyzed enzyme specificity by examining salt (KCl) and magnesium (MgCl2) concentration requirements.
Main Results:
- A DNA endonuclease activity was detected, showing cleavage of UV-damaged DNA without specific preference for thymine dimers.
- Evidence strongly suggests the presence of an apurinic-specific endonuclease, indicated by cleavage at every alkali-labile site in acid-treated DNA.
- The UV- and apurinic-specific activities exhibited distinct salt and magnesium dependencies, highlighting different substrate specificities.
- Osmium tetroxide-treated DNA was also a substrate, with its specific endonuclease activity showing similarities to the UV-acting enzyme.
Conclusions:
- Mouse plasmacytoma cells contain at least two distinct damage-dependent endonuclease activities.
- One activity is specific for apurinic sites, while another acts on UV-induced DNA damage, potentially targeting 5,6-dihydroxydihydrothymine lesions.
- The differential salt and magnesium requirements underscore the distinct nature of these DNA repair enzymes.