RESUMEN
PARP1 rapidly detects DNA strand break damage and allosterically signals break detection to the PARP1 catalytic domain to activate poly(ADP-ribose) production from NAD+. PARP1 activation is characterized by dynamic changes in the structure of a regulatory helical domain (HD); yet, there are limited insights into the specific contributions that the HD makes to PARP1 allostery. Here, we have determined crystal structures of PARP1 in isolated active states that display specific HD conformations. These captured snapshots and biochemical analysis illustrate HD contributions to PARP1 multi-domain and high-affinity interaction with DNA damage, provide novel insights into the mechanics of PARP1 allostery, and indicate how HD active conformations correspond to alterations in the catalytic region that reveal the active site to NAD+. Our work deepens the understanding of PARP1 catalytic activation, the dynamics of the binding site of PARP inhibitor compounds, and the mechanisms regulating PARP1 retention on DNA damage.
Asunto(s)
Daño del ADN , NAD , Dominio Catalítico , Reparación del ADN , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli Adenosina Difosfato Ribosa , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacologíaRESUMEN
Bilokapic at al. (2020) capture PARP2 and its accessory factor HPF1 bridging a DNA break between two nucleosomes, providing a captivating view of the context in which PARP2/HPF1 employ ADP-ribose protein modification to coordinate DNA repair and alter chromatin structure.
Asunto(s)
Cromatina , Poli Adenosina Difosfato Ribosa , Cromatina/genética , Roturas del ADN , Reparación del ADN , Poli(ADP-Ribosa) Polimerasa-1/metabolismoRESUMEN
Catalytic poly(ADP-ribose) production by PARP1 is allosterically activated through interaction with DNA breaks, and PARP inhibitor compounds have the potential to influence PARP1 allostery in addition to preventing catalytic activity. Using the benzimidazole-4-carboxamide pharmacophore present in the first generation PARP1 inhibitor veliparib, a series of 11 derivatives was designed, synthesized, and evaluated as allosteric PARP1 inhibitors, with the premise that bulky substituents would engage the regulatory helical domain (HD) and thereby promote PARP1 retention on DNA breaks. We found that core scaffold modifications could indeed increase PARP1 affinity for DNA; however, the bulk of the modification alone was insufficient to trigger PARP1 allosteric retention on DNA breaks. Rather, compounds eliciting PARP1 retention on DNA breaks were found to be rigidly held in a position that interferes with a specific region of the HD domain, a region that is not targeted by current clinical PARP inhibitors. Collectively, these compounds highlight a unique way to trigger PARP1 retention on DNA breaks and open a path to unveil the pharmacological benefits of such inhibitors with novel properties.
Asunto(s)
Antineoplásicos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Bencimidazoles/farmacología , Reparación del ADN , Roturas del ADN , Daño del ADNRESUMEN
ADP-ribose is a versatile modification that plays a critical role in diverse cellular processes. The addition of this modification is catalyzed by ADP-ribosyltransferases, among which notable poly(ADP-ribose) polymerase (PARP) enzymes are intimately involved in the maintenance of genome integrity. The role of ADP-ribose modifications during DNA damage repair is of significant interest for the proper development of PARP inhibitors targeted toward the treatment of diseases caused by genomic instability. More specifically, inhibitors promoting PARP persistence on DNA lesions, termed PARP "trapping," is considered a desirable characteristic. In this review, we discuss key classes of proteins involved in ADP-ribose signaling (writers, readers, and erasers) with a focus on those involved in the maintenance of genome integrity. An overview of factors that modulate PARP1 and PARP2 persistence at sites of DNA lesions is also discussed. Finally, we clarify aspects of the PARP trapping model in light of recent studies that characterize the kinetics of PARP1 and PARP2 recruitment at sites of lesions. These findings suggest that PARP trapping could be considered as the continuous recruitment of PARP molecules to sites of lesions, rather than the physical stalling of molecules. Recent studies and novel research tools have elevated the level of understanding of ADP-ribosylation, marking a coming-of-age for this interesting modification.
Asunto(s)
Inestabilidad Genómica , Poli(ADP-Ribosa) Polimerasa-1 , Humanos , Adenosina Difosfato Ribosa , Daño del ADN , Reparación del ADN , Poli(ADP-Ribosa) Polimerasa-1/química , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , AnimalesRESUMEN
DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as 'trapping'. To understand the molecular nature of 'trapping' in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.