Combining RNAi and in vivo confocal microscopy analysis of the photoconvertible fluorescent protein Dendra2 to study a DNA repair protein

Gianluca Tell, Matteo Di Piazza, Malgorzata M. Kamocka, Carlo Vascotto

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

Clinical approaches for tumor treatment often rely on combination therapy where a DNA damaging agent is used in combination with a DNA repair protein inhibitor. For this reason, great efforts have been made during the last decade to identify inhibitors of DNA repair proteins or, alternatively, small molecules that specifically alter protein stability or trafficking. Unfortunately, when studying these drug candidates, classical biochemical approaches are prone to artifacts. The apurinic/apyrimidinic endonuclease (APE1) protein is an essential component of the base excision repair (BER) pathway that is responsible for repairing DNA damage caused by oxidative and alkylating agents. In this work, we combined conditional gene expression knockdown of APE1 protein by RNA interference (RNAi) technology with re-expression of an ectopic recombinant form of APE1 fused with the photoconvertible fluorescent protein (PCFP) Dendra2. Dendra2 did not alter the subcellular localization or endonuclease activity of APE1. We calculated APE1 half-life and compared these results with the classical biochemical approach, which is based on cycloheximide (CHX) treatment. In conclusion, we combined RNAi and in vivo confocal microscopy to study a DNA repair protein demonstrating the feasibility and the advantage of this approach for the study of the cellular dynamic of a DNA repair protein.

Original languageEnglish (US)
Pages (from-to)198-203
Number of pages6
JournalBioTechniques
Volume55
Issue number4
DOIs
StatePublished - Oct 1 2013

Keywords

  • APE1/Ref-1
  • Dendra2
  • Live confocal microscopy
  • Protein half-life
  • RNAi

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biotechnology

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