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.
Istituto di Genetica Biochimica ed Evoluzionistica del C.N.R., Pavia, Italy.
A I
AI
Scovassi
C
C
Mariani
M
M
Negroni
C
C
Negri
U
U
Bertazzoni
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism* / Q000494:pharmacology*; D000477:Alkylating Agents / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D001549:Benzamides / Q000494:pharmacology; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D002846:Chromatography, Affinity; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000032:analysis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D015151:Immunoblotting; D008970:Molecular Weight; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000494:pharmacology; D011233:Precipitin Tests; D013463:Sulfuric Acid Esters / Q000494:pharmacology
D000477:Alkylating Agents; D000911:Antibodies, Monoclonal; D001549:Benzamides; D002868:Chromosomal Proteins, Non-Histone; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D013463:Sulfuric Acid Esters; D000246:Adenosine Diphosphate Ribose; C025160:3-aminobenzamide; D011065:Poly(ADP-ribose) Polymerases; D004250:DNA Topoisomerases, Type II; C007482:dimethyl sulfate