The nucleosomal surface is the main target of histone ADP-ribosylation in response to DNA damage

Abstract

ADP-ribosylation is a protein post-translational modification catalyzed by ADP-ribose transferases (ARTs). ART activity is critical in mediating many cellular processes, and is required for DNA damage repair. All five histone proteins are extensively ADP-ribosylated by ARTs upon induction of DNA damage. However, how these modifications aid in repair processes is largely unknown, primarily due to lack of knowledge about where they site-specifically occur on histones. Here, we conduct a comprehensive analysis of histone Asp/Glu ADP-ribosylation sites upon DNA damage induced by dimethyl sulfate (DMS). We also demonstrate that incubation of cell nuclei with NAD+, as has been done previously in the literature, leads to spurious ADP-ribosylation levels of histone proteins. Altogether, we were able to identify 30 modification sites, 20 of which are novel. We also quantify the abundance of these modification sites during the course of DNA damage insult to identify which sites are critical for mediating repair. We found that every quantifiable site increases in abundance over time and that each identified ADP-ribosylation site is located on the surface of the nucleosome. Together, the data suggest specific Asp/Glu residues are unlikely to be critical for DNA damage repair and rather that this process is likely dependent on ADP-ribosylation of the nucleosomal surface in general.

Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1

“…PARylation reactions reconstituted in vitro from purified recombinant components yielded auto-modified PARP1 and PARP2, and also PARylation in trans of other substrates to varying degrees, including core and linker histones. Reactions using nucleosomes prepared from native chromatin could achieve a higher degree of histone PARylation [ 16 , 17 ], suggesting that reactions reconstituted with recombinant components might have been depleted of a key factor. The recently discovered Histone PARylation Factor 1 (HPF1) modulates the enzymatic activity of PARP1 and PARP2 by redirecting their specificity from Asp and Glu to Ser amino acid side chains, and by redirecting both enzymes to more efficiently PARylate histones in trans (in addition to their auto-modification in cis ) [ 18 ].…”

Nonspecific Binding of RNA to PARP1 and PARP2 Does Not Lead to Catalytic Activation

“…We next tested the 18 different RNAs in an activation assay wherein we monitored the incorporation of 32 P-ADPR from 32 P-NAD + into protein using an acid precipitation/ filtration assay. The advantages of this assay compared to smear 29,30 or other gel-based assays 17 are high sensitivity, reproducibility and throughput, allowing us to perform assays under conditions of linearity with respect to product formation (Fig. S4).…”

The multifaceted role of PARP1 in RNA biogenesis

“…Interestingly, the AS patterns provoked by PARP1 KD, differ from those due to PARylation‐inhibition. These results suggest that PARP1's physical association with chromatin structure directly affects splicing decisions, while PARP1 PARylation may regulate splicing indirectly through the PARylation of splicing factors (Ji & Tulin, 2009, 2013) and/or through the PARylation of histones (Karch, Langelier, Pascal, & Garcia, 2017; Yang et al, 2020).…”