Abstract
SummaryADP-ribosylation of proteins is crucial for fundamental cellular processes. Despite increasing examples of DNA ADP-ribosylation, the impact of this modification on DNA metabolism and cell physiology is unknown. Here, we show that the DarTG toxin-antitoxin system from enteropathogenic Escherichia coli (EPEC) catalyzes reversible ADP-ribosylation of single-stranded DNA (ssDNA). The DarT toxin recognizes specific sequence motifs. EPEC DarG abrogates DarT toxicity by two distinct mechanisms: removal of DNA ADP-ribose (ADPr) groups and DarT sequestration. Furthermore, we investigate how cells recognize and deal with DNA ADP-ribosylation. We demonstrate that DNA ADPr stalls replication and is perceived as DNA damage. Removal of ADPr from DNA requires the sequential activity of two DNA repair pathways, with RecF-mediated homologous recombination likely to transfer ADP-ribosylation from single- to double-stranded DNA (dsDNA) and subsequent nucleotide excision repair eliminating the lesion. Our work demonstrates that these DNA repair pathways prevent the genotoxic effects of DNA ADP-ribosylation.
Highlights
ADP-ribosylation is a reversible post-translational modification found in all domains of life (Aravind et al, 2015)
Because of the level of sequence similarity between T. aquaticus and enteropathogenic Escherichia coli (EPEC) DarT (29.5% identity, Figure S1A) and lack of knowledge of its mechanism of action, we assessed whether EPEC DarT can ADP-ribosylate single-stranded DNA (ssDNA)
EPEC DarT was produced by in vitro transcription/ translation (Figure S1B) and incubated with a ssDNA oligonucleotide containing the sequence TCTC and 32P-NAD+ as the co-factor
Summary
ADP-ribosylation is a reversible post-translational modification found in all domains of life (Aravind et al, 2015). Protein ADP-ribosylation has been studied extensively and influences fundamental processes such as transcription, cell division, metabolism, and DNA repair (Barkauskaite et al, 2015; Gibson and Kraus, 2012; Perina et al, 2014). This post-translational modification is catalyzed by mono-ADP-ribosyl transferases (ARTs) or poly-ADP-ribose polymerases (PARPs) that transfer single or multiple ADP-ribose (ADPr) group or groups, respectively, from nicotinamide adenine dinucleotide (NAD+) onto target proteins. Few prokaryotic ARTs have been characterized, most of which are secreted enzymes that contribute to virulence by targeting host proteins. Cholera toxin is secreted by Vibrio cholerae and inactivates G proteins in intestinal epithelial cells (Holmgren et al, 1975), while diphtheria toxin prevents protein translation by ADP-ribosylation of EF-2, leading to host cell death (Strauss and Hendee, 1959)
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