EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions

Marta Rodriguez-Alvarez, Daria Kim, Andriy Khobta

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

The sustainment of replication and transcription of damaged DNA is essential for cell survival under genotoxic stress; however, the damage tolerance of these key cellular functions comes at the expense of fidelity. Thus, translesion DNA synthesis (TLS) over damaged nucleotides is a major source of point mutations found in cancers; whereas erroneous bypass of damage by RNA polymerases may contribute to cancer and other diseases by driving accumulation of proteins with aberrant structure and function in a process termed "transcriptional mutagenesis" (TM). Here, we aimed at the generation of reporters suited for direct detection of miscoding capacities of defined types of DNA modifications during translesion DNA or RNA synthesis in human cells. We performed a systematic phenotypic screen of 25 non-synonymous base substitutions in a DNA sequence encoding a functionally important region of the enhanced green fluorescent protein (EGFP). This led to the identification of four loss-of-fluorescence mutants, in which any ulterior base substitution at the nucleotide affected by the primary mutation leads to the reversal to a functional EGFP. Finally, we incorporated highly mutagenic abasic DNA lesions at the positions of primary mutations and demonstrated a high sensitivity of detection of the mutagenic DNA TLS and TM in this system.

Original languageEnglish
Article number902
Pages (from-to)1-19
Number of pages19
JournalBiomolecules
Volume10
Issue number6
DOIs
Publication statusPublished - 13 Jun 2020

Keywords

  • damage bypass
  • DNA damage
  • DNA damage tolerance
  • enhanced green fluorescent protein (EGFP)
  • host cell reactivation (HCR)
  • mutation assay
  • reporter assay
  • transcriptional mutagenesis
  • translesion synthesis (TLS)

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