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Poly(adenosine diphosphoribose) synthesis in ultraviolet-irradiated xeroderma pigmentosum cells reconstituted with
Abstract:
Synthesis of DNA and poly(adenosine diphosphoribose) [poly(ADPR)] was examined in permeabilized xeroderma pigmentosum lymphoblasts (XP3BE) before and after UV irradiation and in the presence and absence of Micrococcus luteus UV endonuclease. M. luteus UV endonuclease had no effect on the level of DNA or poly(ADPR) synthesis in control, unirradiated cells. UV irradiation caused a decrease in replicative DNA synthesis without any significant change in poly(ADPR) synthesis. In UV-irradiated cells treated with M. luteus UV endonuclease, DNA synthesis was restored to a level slightly greater than in the unirradiated control cells, and poly(ADPR) synthesis increased by 2- to 4-fold. Time--course studies showed that the UV endonuclease dependent poly(ADPR) synthesis preceded the endonuclease-dependent DNA synthesis. Inhibition of endonuclease-dependent poly(ADPR) synthesis with 3-aminobenzamide, 5-methylnicotinamide, or theophylline produced a partial inhibition of the endonuclease-dependent DNA synthesis. Conversely, inhibition of the endonuclease-dependent DNA synthesis with dideoxythymidine triphosphate, phosphonoacetic acid, or aphidicolin had no effect on the endonuclease-dependent poly(ADPR) synthesis. These studies show that stimulation of poly(ADPR) synthesis in UV-irradiated cells occurs subsequent to the DNA strand breaks created by the specific action of the UV endonuclease on UV-irradiated DNA. The effect of the inhibitors of poly(ADPR) synthesis in UV-irradiated cells indicates that the endonuclease-stimulated DNA synthesis is dependent in part on the prior synthesis of poly(ADPR).
Insights
UV irradiation damages DNA, decreasing synthesis. Micrococcus luteus UV endonuclease repairs DNA breaks, restoring synthesis and significantly boosting poly(ADPR) production. This indicates DNA repair synthesis relies on poly(ADPR) synthesis.
Area of Science:
- Molecular Biology
- Biochemistry
- DNA Repair
Background:
- Xeroderma pigmentosum (XP) is a genetic disorder characterized by defective DNA repair.
- UV irradiation induces DNA damage, necessitating efficient repair mechanisms.
- Poly(adenosine diphosphoribose) [poly(ADPR)] is involved in cellular responses to DNA damage.
Purpose of the Study:
- To investigate the interplay between DNA synthesis, poly(ADPR) synthesis, and UV endonuclease activity in XP lymphoblasts.
- To elucidate the role of poly(ADPR) synthesis in the DNA repair process following UV-induced damage.
Main Methods:
- Permeabilized xeroderma pigmentosum lymphoblasts (XP3BE) were used to study DNA and poly(ADPR) synthesis.
- Cells were exposed to UV irradiation with or without Micrococcus luteus UV endonuclease treatment.
- Time-course studies and inhibitor experiments were conducted to assess the sequence and dependency of synthesis pathways.
Main Results:
- UV irradiation reduced DNA synthesis but did not significantly alter poly(ADPR) synthesis.
- Micrococcus luteus UV endonuclease treatment restored DNA synthesis and increased poly(ADPR) synthesis 2- to 4-fold in UV-irradiated cells.
- Poly(ADPR) synthesis preceded UV endonuclease-dependent DNA synthesis, and inhibiting poly(ADPR) synthesis partially reduced DNA synthesis.
Conclusions:
- Stimulation of poly(ADPR) synthesis occurs after UV endonuclease-induced DNA strand breaks.
- Endonuclease-stimulated DNA synthesis is partially dependent on prior poly(ADPR) synthesis, highlighting its crucial role in DNA repair pathways.