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  3. ELTA: Enzymatic Labeling of Terminal ADP-Ribose

ELTA: Enzymatic Labeling of Terminal ADP-Ribose

  • Mol Cell. 2019 Feb 21;73(4):845-856.e5. doi: 10.1016/j.molcel.2018.12.022.
Yoshinari Ando 1,  Elad Elkayam 2,  Robert Lyle McPherson 1,  Morgan Dasovich 1,  Shang-Jung Cheng 1,  Jim Voorneveld 3,  Dmitri V Filippov 3,  Shao-En Ong 4,  Leemor Joshua-Tor 2,  Anthony K L Leung 5
Affiliations

Affiliations

  • 1 Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
  • 2 Keck Structural Biology Laboratory, Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
  • 3 Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, the Netherlands.
  • 4 Department of Pharmacology, University of Washington, Seattle, WA 98195, USA.
  • 5 Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Molecular Biology and Genetics, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA. Electronic address: anthony.leung@jhu.edu.
PMID: 30712989 DOI: 10.1016/j.molcel.2018.12.022
Abstract

ADP-ribosylation refers to the addition of one or more ADP-ribose groups onto proteins. The attached ADP-ribose monomers or Polymers, commonly known as poly(ADP-ribose) (PAR), modulate the activities of the modified substrates or their binding affinities to Other proteins. However, progress in this area is hindered by a lack of tools to investigate this protein modification. Here, we describe a new method named ELTA (enzymatic labeling of terminal ADP-ribose) for labeling free or protein-conjugated ADP-ribose monomers and Polymers at their 2'-OH termini using the Enzyme OAS1 and dATP. When coupled with various dATP analogs (e.g., radioactive, fluorescent, affinity tags), ELTA can be used to explore PAR biology with techniques routinely used to investigate DNA or RNA function. We demonstrate that ELTA enables the biophysical measurements of protein binding to PAR of a defined length, detection of PAR length from proteins and cells, and enrichment of sub-femtomole amounts of ADP-ribosylated Peptides from cell lysates.

Keywords

ADP-ribose; ADP-ribosylated protein; ADP-ribosylation; ADP-ribosyltransferase; enzymatic labeling; mono(ADP-ribosyl)ated protein; oligoadenylate synthetase; poly(ADP-ribose); poly(ADP-ribose) polymerase; poly(ADP-ribosyl)ated protein.

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