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Legionnaires' disease is caused by infection with the intracellularly replicating Gram‐negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (UbADPr) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole‐linked UbADPr, and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked UbADPr. The inhibitory effects of modified Ub on the canonical eukaryotic E1‐enzyme Uba1 are investigated and rationalized in the context of a high‐resolution crystal structure reported herein. Finally, it is shown that synthetic UbADPr analogues can be used to effectively pull‐down overexpressed DupA from cell lysate. [ABSTRACT FROM AUTHOR]

ADP-ribosylation (ADPr) is a widespread post-translational modification (PTM) spanning all kingdoms of life. It is employed by bacteria and viruses in their war against the host, and by eukaryotes to regulate many physiological processes, across almost all cellular compartments. PARP1, the founding member of the PARP family, is an early sensor of single- and double-strand breaks and catalyzes ADPr to mediate DNA damage repair. The recent discovery of Serine-ADPr and the PARP1 accessory factor HPF1 has brought a momentous change to the field. Bolstered by innovative ways to study ADPr, new and exciting research directions are rapidly emerging. In this review we explore our understanding of the HPF1/PARP1-mediated ADPr signaling pathway in DNA damage. We focus on the mechanistic steps leading to Serine-ADPr and its relevance in the DNA damage response. We discuss important technological advances that have enabled a nuanced study of Serine-ADPr, and conclude with an overview of the role of PARP inhibitors in cancer therapy. • Serine-ADPr is a widespread PTM in DNA damage, synthesized by the HPF1/PARP1 complex. • Serine-ADPr removal is mediated through ARH3, a protein associated with a neurodegenerative disease. • Many tools have been developed for the study of Ser-ADPr, including ADPribosylated peptides, ADPr-binding reagents and antibodies. • The main functional consequences of Ser-ADPr are protein recruitment to sites of DNA damage, chromatin relaxation, and PTM interplay. • PARP1 inhibition is used clinically for cancer treatment. The discovery of Ser-ADPr and HPF1 opens up new avenues in drug discovery. [ABSTRACT FROM AUTHOR]