ADP-ribosylation of nonhistone proteins in HeLa cells: modification of DNA topoisomerase II.
We have studied the nonhistone proteins which are modified by ADP-ribosylation in HeLa cells. When isolated nuclei were incubated with 32P-NAD, the main labeled proteins presented sizes of 170, 116, 70, and 45 kDa. To provide evidence for the identification of the 170-kDa band as DNA topoisomerase II, the enzyme was immunoprecipitated from isolated nuclei incubated with 32P-NAD and a labeled peptide of 170 kDa was observed. The label was sensitive to the action of venom phosphodiesterase which specifically degrades ADP-ribose. ADP-ribosylated proteins were also isolated from HeLa cells by affinity chromatography on boronate-agarose gel. Using a monoclonal antibody against the 170-kDa isoform of topoisomerase II, a single 170-kDa immunoreactive peptide was recognized by Western blot among the retained protein acceptors. When ADP-ribosylation was blocked by treating HeLa cells with 3-aminobenzamide, topoisomerase II was no longer retained on the boronate column. These results provide experimental evidence indicating that DNA topoisomerase II is ADP-ribosylated in HeLa cells. To possibly correlate ADP-ribosylation of nuclear proteins with the extent of DNA damage, permeabilized HeLa cells were incubated with 32P-NAD after treatment with the alkylating agent dimethylsulfate. ADP-ribosylated proteins were isolated by boronate chromatography. A strong increase in the ADP-ribosylation of the poly(ADP-ribose)polymerase was observed, whereas no further modification of topoisomerase II was noted.[1]References
- ADP-ribosylation of nonhistone proteins in HeLa cells: modification of DNA topoisomerase II. Scovassi, A.I., Mariani, C., Negroni, M., Negri, C., Bertazzoni, U. Exp. Cell Res. (1993) [Pubmed]
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