The establishment of methods for free PAR generation and PAR reader detection

Yueshuang Ke; Ke Wang; Hui Xu; Chenxin Wang; Jing Zhang; Xianlu Zeng; Ruoxi Wang; Istvan Boldogh; Xueqing Ba

doi:10.1016/j.mcp.2018.04.002

The establishment of methods for free PAR generation and PAR reader detection

Mol Cell Probes. 2018 Jun:39:57-60. doi: 10.1016/j.mcp.2018.04.002. Epub 2018 Apr 5.

Authors

Yueshuang Ke¹, Ke Wang², Hui Xu³, Chenxin Wang², Jing Zhang², Xianlu Zeng¹, Ruoxi Wang², Istvan Boldogh⁴, Xueqing Ba⁵

Affiliations

¹ The Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024, China; School of Life Science, Northeast Normal University, Changchun, Jilin 130024, China.
² School of Life Science, Northeast Normal University, Changchun, Jilin 130024, China.
³ School of Physical Education, Northeast Normal University, Changchun, Jilin 130024, China.
⁴ Department of Microbiology and Immunology, Sealy Center for Molecular Medicine, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA.
⁵ The Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024, China; School of Life Science, Northeast Normal University, Changchun, Jilin 130024, China. Electronic address: baxq755@nenu.edu.cn.

PMID: 29627626
DOI: 10.1016/j.mcp.2018.04.002

Abstract

Poly (ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor that catalyzes the poly (ADP-ribose) (PAR) onto a variety of target proteins, such as histones, DSB repair factors and PARP1 itself under consumption of NAD⁺. Besides, PARP1 can affect a variety of proteins in noncovalent modification manner to carry out specific cellular functions. Here, we established a method to generate non-radiolabeled free PAR by PARG moderately cleaving PAR from autoPARylated PARP1, and utilized dot-blot assay to determine the interaction between free PAR and interested proteins. The methods to generate free PAR and detect the noncovalent interactions between proteins and free PAR are nonradioactive and convenient, which will facilitate the studies to explore the significance of PAR reading in various biological processes.

Keywords: Dot-blot assay; Free PAR; Noncovalent; PARG; PARP1.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

ELAV-Like Protein 1 / metabolism
Humans
Molecular Probe Techniques*
Poly Adenosine Diphosphate Ribose / analysis*
Recombinant Fusion Proteins / isolation & purification

Substances

ELAV-Like Protein 1
Recombinant Fusion Proteins
Poly Adenosine Diphosphate Ribose