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Analysis of DNA Processing Enzyme FEN1 and Its Regulation by Protein Lysine Acetylation

  • Onyekachi E. Ononye
  • Catherine W. Njeri
  • Lata BalakrishnanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1983)

Abstract

Cellular proteins are modified by lysine acetylation wherein an acetyltransferase transfers an acetyl group from acetyl co enzyme A onto the e-amino group of lysine residues. This modification is extremely dynamic and can be reversed by a deacetylase that removes the acetyl group. Addition of acetyl group to the lysine residue neutralizes its positive charge, thereby functioning as a molecular switch in regulating the enzymatic functions of the protein, its stability, and it cellular localization. Since this modification is extremely dynamic within the cell, biochemical studies characterizing changes in protein function are imperative to understand how this modification alters protein function in a specific cellular pathway. This unit describes in detail expression and purification of a recombinant nuclease and acetyltransferase, in vitro acetylation of the recombinant protein and biochemical assays to study the changes in enzymatic activity of the in vitro acetylated nuclease.

Keywords

Lysine acetylation In vitro acetylation Lysine acetyltransferase p300 Flap endonuclease 1 (FEN1) Nuclease enzyme assays 

Notes

Acknowledgments

This work was supported by NIH Grant GM0938328 and New Faculty Start-Up Funds from Indiana University Purdue University Indianapolis (IUPUI).

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Copyright information

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

Authors and Affiliations

  • Onyekachi E. Ononye
    • 1
  • Catherine W. Njeri
    • 1
  • Lata Balakrishnan
    • 1
    Email author
  1. 1.Department of Biology, School of ScienceIndiana University Purdue University IndianapolisIndianapolisUSA

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