Shelterin/telosome is a multi-protein complex at mammalian telomeres, anchored to the double-stranded region by the telomeric-repeat binding factors-1 and -2. In vitro modification of these proteins by poly(ADP-ribosyl)ation through poly(ADP-ribose) polymerases-5 (tankyrases) and -1/-2, respectively, impairs binding. Thereafter, at least telomeric-repeat binding factor-1 is degraded by the proteasome. We show that pharmacological inhibition of poly(ADP-ribose) polymerase activity in cells from two different species leads to rapid decrease in median telomere length and stabilization at a lower setting. Specific knockdown of poly(ADP-ribose) polymerase-1 by RNA interference had the same effect. The length of the single-stranded telomeric overhang as well as telomerase activity were not affected. Release of inhibition led to a fast re-gain in telomere length to control levels in cells expressing active telomerase. We conclude that poly(ADP-ribose) polymerase-1 activity and probably its interplay with telomeric-repeat binding factor-2 is an important determinant in telomere regulation. Our findings reinforce the link between poly(ADP-ribosyl)ation and aging/longevity and also impact on the use of poly(ADP-ribose) polymerase inhibitors in tumor therapy.
Poly(ADP‐ribose) (PAR) is a complex and reversible post‐translational modification that controls protein function and localization through covalent modification of, or noncovalent binding to target proteins. Previously, we and others characterized the noncovalent, high‐affinity binding of the key nucleotide excision repair (NER) protein XPA to PAR. In the present study, we address the functional relevance of this interaction. First, we confirm that pharmacological inhibition of cellular poly(ADP‐ribosyl)ation (PARylation) impairs NER efficacy. Second, we demonstrate that the XPA–PAR interaction is mediated by specific basic amino acids within a highly conserved PAR‐binding motif, which overlaps the DNA damage‐binding protein 2 (DDB2) and transcription factor II H (TFIIH) interaction domains of XPA. Third, biochemical studies reveal a mutual regulation of PARP1 and XPA functions showing that, on the one hand, the XPA–PAR interaction lowers the DNA binding affinity of XPA, whereas, on the other hand, XPA itself strongly stimulates PARP1 enzymatic activity. Fourth, microirradiation experiments in U2OS cells demonstrate that PARP inhibition alters the recruitment properties of XPA‐green fluorescent protein to sites of laser‐induced DNA damage. In conclusion, our results reveal that XPA and PARP1 regulate each other in a reciprocal and PAR‐dependent manner, potentially acting as a fine‐tuning mechanism for the spatio‐temporal regulation of the two factors during NER.
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