Figure 5: PARP3 binds nicked nucleosomes and ribosylates H2BE2.
From: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2
(a) ADP-ribosylation of E2 in recombinant H2B by hPARP3. MS/MS fragmentation profile of doubly charged PE(+15)PAKSAPAPK (theoretical: 554.3115 m/z; observed: 554.3113 m/z; 0.4 p.p.m. mass error), indicating the position of the NH (+15.0109 Da) moiety on glutamate resulting from hydroxylamine derivatization of ADP-ribose. Preferred fragmentation N-terminal to prolines is observed, as expected43. (b) Mutation of E2 reduces ribosylation of H2B by hPARP3. The products of ribosylation reactions containing 200 nM hPARP3, 12.5 μM biotin-NAD+, 100 nM nicked oligonucleotide duplex (50 bp), and 5 μM of wild-type or mutant recombinant H2B were separated on 15% SDS–PAGE gels and detected by autoradiaography. (c) PARP3 binds to nicked mononuclesomes. (left) Reconstituted mononucleosomes were assembled on intact or nicked DNA (Widom positioning sequence 601.2) and nucleosome quality assessed by native gel electrophoresis. (right) representative images of negative stained intact (top) or nicked (bottom) nucleosomes incubated with His-tagged cPARP3, and with the His-tagged protein detected by nanogold Ni-NTA. Scale bars, 50nm. (d) PARP3 ribosylates H2BE2 in reconstituted nicked mononucleosomes. Hundred nanomolar of intact or nicked 601.2 DNA, present either as naked duplex or within reconstituted nucleosomes containing wild-type or mutant H2B, was incubated with 100 nM hPARP1 (lanes 1–6) or hPARP3 (lanes 7–12) and either 12.5 μM biotin-NAD+ (hPARP3) or 1.5 μM biotin-NAD+ (hPARP1, to encourage shorter chain modifications). MS/MS, tandem mass spectrometry.
