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Reversible protein phosphorylation modulates nucleotide excision repair of damaged DNA by human cell extracts
R R Ariza1, S M Keyse, J G Moggs
1Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, UK.
Abstract:
Nucleotide excision repair of DNA in mammalian cells uses more than 20 polypeptides to remove DNA lesions caused by UV light and other mutagens. To investigate whether reversible protein phosphorylation can significantly modulate this repair mechanism we studied the effect of specific inhibitors of Ser/Thr protein phosphatases. The ability of HeLa cell extracts to carry out nucleotide excision repair in vitro was highly sensitive to three toxins (okadaic acid, microcystin-LR and tautomycin), which block PP1- and PP2A-type phosphatases. Repair was more sensitive to okadaic acid than to tautomycin, suggesting the involvement of a PP2A-type enzyme, and was insensitive to inhibitor-2, which exclusively inhibits PP1-type enzymes. In a repair synthesis assay the toxins gave 70% inhibition of activity. Full activity could be restored to toxin-inhibited extracts by addition of purified PP2A, but not PP1. The p34 subunit of replication protein A was hyperphosphorylated in cell extracts in the presence of phosphatase inhibitors, but we found no evidence that this affected repair. In a coupled incision/synthesis repair assay okadaic acid decreased the production of incision intermediates in the repair reaction. The formation of 25-30mer oligonucleotides by dual incision during repair was also inhibited by okadaic acid and inhibition could be reversed with PP2A. Thus Ser/Thr- specific protein phosphorylation plays an important role in the modulation of nucleotide excision repair in vitro.
Insights
Protein phosphorylation regulates DNA repair. Inhibiting protein phosphatases like PP2A significantly impairs nucleotide excision repair in mammalian cells, affecting DNA lesion removal.
Area of Science:
- Molecular Biology
- Biochemistry
- DNA Repair Mechanisms
Background:
- Mammalian DNA repair involves over 20 proteins to remove UV-induced and other mutagenic DNA lesions.
- The role of reversible protein phosphorylation in modulating DNA repair pathways remains an area of investigation.
Purpose of the Study:
- To investigate the impact of Ser/Thr protein phosphatase inhibition on nucleotide excision repair (NER) in vitro.
- To determine if protein phosphorylation significantly modulates the efficiency of DNA repair mechanisms.
Main Methods:
- Utilized HeLa cell extracts to perform in vitro nucleotide excision repair assays.
- Employed specific Ser/Thr protein phosphatase inhibitors: okadaic acid, microcystin-LR, and tautomycin.
- Assessed repair synthesis and coupled incision/synthesis activities in the presence of inhibitors and purified phosphatases.
Main Results:
- Nucleotide excision repair activity was highly sensitive to okadaic acid and microcystin-LR, indicating involvement of PP2A-type phosphatases.
- Toxins inhibited repair synthesis by up to 70%, with full activity restored by adding purified PP2A.
- Okadaic acid reduced incision intermediates and the formation of 25-30mer oligonucleotides, demonstrating impaired dual incision.
Conclusions:
- Serine/threonine-specific protein phosphorylation plays a crucial role in modulating nucleotide excision repair in vitro.
- PP2A-type phosphatases are key regulators of the DNA repair process, influencing incision steps.
- Reversible protein phosphorylation is a significant regulatory mechanism for DNA repair efficiency in mammalian cells.