FIGURE 1.
PARP1 is a PIASy-mediated SUMOylated protein on mitotic chromosomes. A, PARP1 is SUMOylated on mitotic chromosomes. Chromosomal fractions were prepared in CSF extracts in the presence of either dnUbc9 or PIASy as described under “Experimental Procedures.” Isolated chromosomes were analyzed by immunoblotting with indicated antibodies. Arrows indicate the position of SUMOylated PARP1, which were diminished when SUMOylation was inhibited by dnUbc9. B, PIASy is required for PARP1 SUMOylation. CSF extracts were subjected to immunodepletion with anti-PIASy antibody (−PIASy), and then a purified recombinant PIASy was supplemented to PIASy-depleted extracts (−PIASy+Rec.PIASy). Chromosomal fractions were prepared in the indicated conditions of CSF extracts, then the SUMOylation status of isolated chromosomes was analyzed with the indicated antibodies. Arrows indicate the position of SUMOylated PARP1. C, PARP1 SUMOylation is specific in mitosis. CSF extracts were released into interphase by addition of CaCl2. Sperm nuclei were added to either CSF or interphase extracts. After incubation of the indicated periods with interphase extracts or 40 min with CSF extracts, the chromatin fractions were isolated as described under “Experimental Procedures.” Isolated chromatin fractions were subjected to immunoblotting with the indicated antibodies. Arrows indicate SUMOylated PARP1. D, representation of functional domains of PARP1 (adjusted from Ref. 18) is shown. Lines indicate the fragments of PARP1 that were used as antigens. BRCT, BRCA1 C-terminal domain.