An HPF1/PARP1-Based Chemical Biology Strategy for Exploring ADP-Ribosylation

HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation

“…Consistent with this, the immunodepletion of PARP1 or HPF1 attenuated LIG3-XRCC1 function in Okazaki fragment joining. Our findings are in agreement with a recent report that PARP1 and histone H3 are predominantly mono-ADP-ribosylated upon DNA damage in an HPF1/PARP1-dependent manner ( 54 ). Our data suggest that LIG3-mediated ligation of Okazaki fragments does not depend on the PAR binding activity of XRCC1.…”

Uncovering the Invisible: Mono-ADP-ribosylation Moved into the Spotlight

“…Reduced NAD + levels following ARTD1-mediated hyper-consumption might even affect ARTD1 itself. This possibility is in agreement with recent reports, suggesting that even upon DNA damage, ARTD1 predominantly catalyzes protein MARylation rather than PARylation [148]. Interestingly, overexpression of ARTD10 that led to enhanced mono-ADP-ribosylation was sufficient to repress cellular NAD + levels and, consequently, the MARylating activities of ARTD14, ARTD10, ARTD12 and ARTD8 [107].…”

“…Nonetheless, while the new antibodies recognize MAR and PAR and therefore are referred to as pan-ADP-ribose antibodies, they could already be used to study organelle and mono-ART-specific ADP-ribosylation dynamics by Western blotting (WB) and Immunofluorescence (IF) in different contexts [127,175]. A recent study finally reported the generation of several MARylation-specific antibodies that did not cross-react with PAR [148]. The peptides used to generate these antibodies were ADP-ribosylated in vitro, and enzyme promiscuity was prevented by chemically protecting alternative ADPr amino acid acceptor sites.…”

The regulatory landscape of the human HPF1- and ARH3-dependent ADP-ribosylome

“…The enrichment of ADPr-modified peptides without downstream PARG treatment furthermore allowed us to infer the cellular MARylation versus PARylation equilibrium, hereby uncovering that MARylation is a global and dominant phenomenon in human cells. While observations capture the state of the cell upon lysis, and considering that ADPr is a very dynamic modification (Polo and Jackson, 2011), the detected MARylation probably stems from substrates initially being modified with PAR, which is then rapidly degraded in vivo into MAR by high hydrolase activity of eraser enzymes (Bonfiglio et al, 2020; Fontana et al, 2017; Hanzlikova et al, 2020), and PARP1 itself (Rudolph et al, 2020). Still, considering this dynamic regulation of ADPr has remained unnoticed for decades, along with the importance of better understanding the endogenous ADPr equilibrium, underscores the necessity for investigating ADPr dynamics under native conditions using unbiased proteomics strategies.…”

“…The catalytic activity of HPF1-PARP1/2 is counteracted by poly-ADP-ribose glycohydrolase (PARG) (Slade et al, 2011), and ADP-ribosylhydrolase 3 (ARH3) (Abplanalp et al, 2017; Fontana et al, 2017), providing an additional layer of complexity to the cellular ADPr dynamics. This leads to high turnover of poly-ADP-ribosylation (PAR) and certain PARP1-catalyzed modifications existing as mono-ADPr (MAR) events in human cells (Bonfiglio et al, 2020; Fontana et al, 2017). With PARP1 described as a poly-ADPr (PAR) polymerase (Kraus, 2015; Schreiber et al, 2006), the notion that PARP1 substrates primarily exist as MARylated entails fundamental biological insights and epitomizes the relevance of exploring the modularity of ADPr under in vivo conditions.…”