Research Article

Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation

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Science  01 Jul 2016:
Vol. 353, Issue 6294, pp. 45-50
DOI: 10.1126/science.aaf7865

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Mapping ADP-ribosylation by PARPs

During many cell processes, ADP-ribose is transferred from NAD+ onto protein substrates by poly(ADP-ribose) polymerases (PARPs). Gibson et al. developed a method to track ribose transfer events and mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome and genome. One PARP-1 target is NELF, a protein complex that regulates pausing by RNA polymerase II. If NELF is ribosylated, pausing is released and productive transcription elongation resumes.

Science, this issue p. 45

Abstract

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.

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