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Biochemical and Cellular Assays to Assess the Effects of Acetylation on Base Excision Repair Enzymes

  • Shrabasti Roychoudhury
  • Suravi Pramanik
  • Hannah L. Harris
  • Kishor K. BhakatEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1983)

Abstract

Protein posttranslational modifications (PTMs), including acetylation, have emerged as important regulators for controlling many cellular processes. DNA base excision repair (BER), a highly coordinated multistep cellular process, is primarily involved in the repair of both endogenous and drug-induced exogenous DNA base damages. BER relies on sequential recruitment and coordinated actions of multiple proteins. Increasing evidence suggests that acetylation of lysine residues of BER proteins facilitates fine-tuning of enzymatic activities, protein-protein interactions, and coordination of the steps in BER pathway. In this chapter, we describe detailed in vitro and in vivo approaches to examine the effect of acetylation on BER enzymes, focusing on the impact of acetylation of AP-endonuclease (APE1), a key enzyme in BER pathway, on its DNA damage repair activity, substrate-binding, and subcellular localization.

Keywords

Acetylation BER DNA damage APE1 Comet assay EMSA In vitro acetylation Colony formation assay DNA repair assay 

Notes

Acknowledgment

This research was supported by NIH/NCI R01CA148941 and Nebraska state DHS LB506 grants.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Shrabasti Roychoudhury
    • 1
  • Suravi Pramanik
    • 1
  • Hannah L. Harris
    • 1
  • Kishor K. Bhakat
    • 1
    • 2
    Email author
  1. 1.Department of Genetics, Cell Biology and AnatomyUniversity of Nebraska Medical CenterOmahaUSA
  2. 2.Fred and Pamela Buffet Cancer CenterUniversity of Nebraska Medical CenterOmahaUSA

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