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Repair of potentially lethal damage by introduction of T4 DNA ligase in eucaryotic cells
M Durante1, G F Grossi, M Napolitano
1Dipartimento di Scienze Fisiche, Università di Napoli, Italy.
Abstract:
The bacterial enzyme PvuII, which generates blunt-ended DNA double-strand breaks, and T4 DNA ligase, which seals adjacent DNA fragments in coupling to ATP cleavage, were introduced in mouse C3H10T1/2 fibroblasts using osmolytic shock of pinocytic vesicles. Cells were then assayed for their clonogenic ability. In agreement with previous studies by others, we find that the PvuII restriction endonuclease simulates ionizing radiation effects by causing a dose-dependent loss of reproductive capacity. Here we show that the concomitant treatment with DNA ligase considerably increases cell survival. Survival curves were shown to be dependent on the ligase enzyme dose and on ATP concentration in the hypertonic medium. We conclude that T4 DNA ligase is able to repair some of the potentially lethal damage produced by restriction endonucleases in eucaryotic cells.
Insights
The bacterial enzyme PvuII causes DNA damage similar to radiation, reducing cell survival. However, T4 DNA ligase treatment significantly enhances cell survival by repairing this damage in eukaryotic cells.
Area of Science:
- Molecular biology
- Cell biology
- Biochemistry
Background:
- Restriction endonucleases like PvuII induce DNA double-strand breaks.
- DNA ligase enzymes are crucial for DNA repair and ligation.
Purpose of the Study:
- To investigate the effects of PvuII endonuclease and T4 DNA ligase on the survival of mouse fibroblasts.
- To determine if T4 DNA ligase can repair DNA damage induced by PvuII in eukaryotic cells.
Main Methods:
- Mouse C3H10T1/2 fibroblasts were treated with PvuII endonuclease and T4 DNA ligase via osmolytic shock.
- Cellular clonogenic ability was assayed to measure survival rates.
- Survival curves were analyzed based on enzyme and ATP concentrations.
Main Results:
- PvuII treatment caused a dose-dependent decrease in cell reproductive capacity, mimicking ionizing radiation.
- Concomitant treatment with T4 DNA ligase significantly increased cell survival.
- Enhanced survival was dependent on both T4 DNA ligase dose and ATP concentration.
Conclusions:
- T4 DNA ligase can repair potentially lethal DNA damage induced by restriction endonucleases in eukaryotic cells.
- This study highlights the potential of DNA ligase as a therapeutic agent against DNA-damaging agents.