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

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.

Back-Up Base Excision DNA Repair in Human Cells Deficient in the Major AP Endonuclease, APE1

“…Systems based on plasmids with lesions introduced into a reporter gene are valuable tools for studying repair in living cells. In one particularly useful design, a lesion is placed in a dysfunctional reporter and detects the level of transcriptional mutagenesis (TM, misincorporation of NMPs opposite the lesion by an RNA polymerase) that restores the activity [ 51 , 61 ]. For example, a c.613C > T mutation in the eGFP gene replaces the 5′-CAG-3′ Gln205 codon with the 5′-TAG-3′ stop codon, leading to a loss of fluorescence, while any amino acid potentially arising from the TAG codon by a single-nucleotide substitution (Gln, Glu, Leu, Lys, Ser, Trp, or Tyr) produces fluorescent protein ( Figure 1 a).…”

“…The eGFP coding sequence conveniently has two nickase sites near each other, making it possible to introduce any lesion between them by oligonucleotide replacement [ 57 ]. Several nonsense and missense mutations in this region that result in non-fluorescent EGFP have been identified [ 51 , 57 , 61 ]. In the presence of DNA damage, repair converts the eGFP sequence back to coding the non-fluorescent form; on the contrary, lack of repair is manifested in the appearance of fluorescing cells.…”

“…In the presence of DNA damage, repair converts the eGFP sequence back to coding the non-fluorescent form; on the contrary, lack of repair is manifested in the appearance of fluorescing cells. The assay was successfully applied to analyze the repair of 8-oxoG, U, thymine glycol, AP sites, and single-strand breaks in cellulo [ 51 , 61 , 65 , 86 , 87 , 88 , 89 ]. Since the TM outcome is strongly influenced by the ability of RNA polymerase II to bypass the lesion and by the accompanying misincorporation spectrum, the TM assay is best suited for quantitative comparisons of the same lesions under the conditions of different repair capacities, which can be modulated either by the host cell genotype or by small lesion modifications affecting their removal by DNA repair.…”

Transcriptional mutagenesis-based reporter system for the analysis of DNA repair

“…Methods and Algorithms: The knockout of BER genes APEX1, OGG1, and MUTYH were generated using genome editing technology CRISPR/Cas9 from the parental 293FT cells. To detect the DNA repair in cells we use the assay based on plasmids containing single nucleotide substitution in the EGFP gene, which results in a loss of fluorescence [2]. The lesion is introduced in the position of the substitution.…”

DNA Damage Response

“…In addition, mammalian DNA-dependent RNA polymerases are also able to introduce mistakes into the RNA structure and, thus, result in modified proteins via “transcriptional mutagenesis”. Rodriguez-Alvarez et al [ 16 ] developed and presented a new technology in order to detect TLS. In particular, the authors present the enhanced green fluorescent protein (EGFP)-based reporter that can be used for the direct and sensitive detection of mutagenic DNA lesions bypasses.…”