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

Marta Rodriguez-Alvarez,Andriy Khobta,Daria Kim

doi:10.3390/biom10060902

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.

Highlights

Damage to DNA is usually repaired in cells within minutes; repair efficiency can be limited by a number of factors
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)
Its DNA sequence allows for the strand-specific incorporation of oligonucleotides containing synthetic DNA lesions at defined positions and, especially important, any single nucleotide substitution at the position 613 of the coding sequence leads to an amino acid which reconstitutes the enhanced green fluorescent protein (EGFP) fluorescence [30]

Summary

Introduction

Damage to DNA is usually repaired in cells within minutes; repair efficiency can be limited by a number of factors (for instance, peculiar structure of DNA modifications, their accessibility to DNA repair proteins, oversaturation of the cellular repair capacity by excessive damage loads, or impairment of the relevant repair pathway by a genetic, epigenetic or pharmacological mechanism). One of the most important DNA damage tolerance mechanisms is the ability of DNA polymerases to bypass DNA damage during replication [3]. As replicative DNA polymerases have very low capacities of DNA synthesis past the damaged bases, dedicated translesion synthesis (TLS) enzymes are required for copying damaged DNA [4,5]. Since TLS capacity is incompatible with proofreading exonuclease activity, all DNA polymerases specialized in damage bypass have elevated error rates. This makes TLS an important source of DNA damage-induced mutagenesis and the major cause of point mutations found in cancers [6,7]

Methods

Results

Discussion

Conclusion