PARP1 Writes N3-Cytidine ADP-Ribosylation in DNA

Recent evidence indicates that mono - and poly-ADP ribosylation (MARylation and PARylation) are not limited to proteins but extend to DNA. Notably, in vitro base PARylation by PARP1 in single stranded DNA (ssDNA) was demonstrated at N1-deoxyadenosine (N1-dA). Here, we report that PARP1 catalyzes N3-specific ADP-ribosylation of deoxycytidine (N3-dC) in single-stranded DNA. Analogous to N1-dA PARylation, which is prone to spontaneous adenine-to-inosine deamination, N3-dC PARylation promotes cytosine deamination, yielding N3-PARylated-deoxyuridine. These deamination products yield diagnostic PARylation signatures in LC-MS/MS, namely N1-ribosyl-deoxyinosine (N1-r-dI) and N3-ribosyl-deoxyuridine (N3-r-dU). We synthesized both N1-r-dI and N3-r-dU as diagnostic standards and established absolute quantification of base ADP-ribosylations by LC-MS/MS. Quantitative analysis of PARylated dA and dC in ssDNA reveals pronounced sequence preferences of PARP1. Removal of these base modifications differs markedly, since ADP-ribose glycohydrolase TARG1 removes PAR from both dA and dC, whereas PARG acts exclusively on dA. Our results establish cytidine ADP-ribosylation as a novel DNA modification, with potential roles in DNA metabolism, epigenetic regulation, or genome stability.