Figure 2: Mechanism of DNA break binding by the cPARP3 WGR domain.
From: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2
(a) Schematic representation of PARP3 domains with the human (top) and chicken (bottom) amino acid positions indicated. The WGR domain is shown in expanded format below showing the HSQC perturbed residues of chicken PARP3 (bottom) and the equivalent residues in human PARP3 (top). (b) Overlay of 1H–15N HSQC NMR full spectra for cPARP31–169 in the absence (blue) or presence (red) of oligodeoxyribonucleotide duplex harbouring a 5′-phosphorylated nick (protein:DNA ratios of 1:0 and 1:2, respectively). (c) Expanded view of the small boxed region shown in b demonstrating the chemical shifts induced in cPARP31–169 by different concentrations of nicked DNA. Protein:DNA ratios were 1:0 (that is, no DNA; blue), 5:1 (magenta), 1:1 (green) and 1:2 (red). (d) Map of significant chemical shifts induced in cPARP31–169 by DNA duplex harbouring a 5′-phosphorylated nick (>0.1 p.p.m) or 10-bp 3′-overhang with a recessed 5′-phosphorylated terminus (>0.04), surface modelled using CS-Rosetta41. Residues with a significant chemical shift in the presence of either a nick (blue) or 3′-overhang (green) or both (red) are indicated. (e) Electrostatic surface of modelled cPARP31–169 with nicked DNA. (f) Model of cPARP31–169 with nicked DNA, depicting residues with significant chemical shifts as above. (g) Model of cPARP31–169 with nicked DNA lacking the strand located upstream (5′) of the nick (that is, harbouring a DSB with 10-bp 3′-overhang). Residues exhibiting a significant chemical shift are indicated as above.
