Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells

Abstract

Genome integrity is constantly threatened by DNA lesions arising from numerous exogenous and endogenous sources. Survival depends on immediate recognition of these lesions and rapid recruitment of repair factors. Using laser microirradiation and live cell microscopy we found that the DNA-damage dependent poly(ADP-ribose) polymerases (PARP) PARP-1 and PARP-2 are recruited to DNA damage sites, however, with different kinetics and roles. With specific PARP inhibitors and mutations, we could show that the initial recruitment of PARP-1 is mediated by the DNA-binding domain. PARP-1 activation and localized poly(ADP-ribose) synthesis then generates binding sites for a second wave of PARP-1 recruitment and for the rapid accumulation of the loading platform XRCC1 at repair sites. Further PARP-1 poly(ADP-ribosyl)ation eventually initiates the release of PARP-1. We conclude that feedback regulated recruitment of PARP-1 and concomitant local poly(ADP-ribosyl)ation at DNA lesions amplifies a signal for rapid recruitment of repair factors enabling efficient restoration of genome integrity.

Simultaneous dual-channel imaging to quantify interdependent protein recruitment to laser-induced DNA damage sites

“…Thus we compared early recruitment dynamics of PARP1 and ALC1, both of which are known as fast recruiters with maximum recruitment reached within < 1 min [15–17,29,48,49] (Figure 7). …”

“…To substantiate the dependence of ALC1 recruitment on PARP1 catalytic activity, we repeated the experiments in PARP1 WT and KO cells using a catalytic mutant of PARP1, E988K, which shows slower accumulation and longer residence at DNA damage sites [17,51] (Supplementary Figure 8). In PARP1 WT cells, ALC1-mEGFP or ALC1-mCherry showed slower recruitment when co-transfected with PARP1 E988K compared to PARP1 WT (Figure 7b, d, f, h).…”

“…PARP1 is rapidly and transiently recruited to DNA damage sites reaching a maximum about 1 minute on average after microirradiation [17]. Its initial recruitment is mediated by the Zn-finger DNA-binding domain and by PAR-binding, while its dissociation is dependent on its catalytic activity [17].…”

“…Its initial recruitment is mediated by the Zn-finger DNA-binding domain and by PAR-binding, while its dissociation is dependent on its catalytic activity [17]. DNA-damage dependent activation of PARP1 represents an early response to genotoxic stress with PARP1 often referred to as the sensor of DNA strand breaks [18].…”

“…PARP1 mediates the initial accumulation of MRE11–RAD50–NBS1 (MRN) complex at DNA lesions and promotes rapid recruitment of BRCA1, BRCA2 and EXO1 involved in homologous recombination [26–29]. PAR also promotes recruitment of XRCC1 as a scaffold protein in base excision repair [17,30,31] and XPA as a scaffold protein in nucleotide excision repair [32]. …”