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[Research Article] Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation

ScienceNOW - Fetched: June 30th, 2016, 3:00pm UTC

Poly[adenosine diphosphate (ADP)–ribose] polymerases (PARPs) are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from the oxidized form of nicotinamide adenine dinucleotide (NAD+) onto substrate proteins. Here we report a robust NAD+ analog–sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent copper-catalyzed azide-alkyne cycloaddition reactions. Using this approach, we mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome, as well as thousands of PARP-1–mediated ADP-ribosylation sites across the genome. We found that PARP-1 ADP-ribosylates and inhibits negative elongation factor (NELF), a protein complex that regulates promoter-proximal pausing by RNA polymerase II (Pol II). Depletion or inhibition of PARP-1 or mutation of the ADP-ribosylation sites on NELF-E promotes Pol II pausing, providing a clear functional link between PARP-1, ADP-ribosylation, and NELF. This analog-sensitive approach should be broadly applicable across the PARP family and has the potential to illuminate the ADP-ribosylated proteome and the molecular mechanisms used by individual PARPs to mediate their responses to cellular signals. Authors: Bryan A. Gibson, Yajie Zhang, Hong Jiang, Kristine M. Hussey, Jonathan H. Shrimp, Hening Lin, Frank Schwede, Yonghao Yu, W. Lee Kraus

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