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

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 p...

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Detalles Bibliográficos
Autores principales: Rodriguez-Alvarez, Marta, Kim, Daria, Khobta, Andriy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357151/
https://www.ncbi.nlm.nih.gov/pubmed/32545792
http://dx.doi.org/10.3390/biom10060902
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author Rodriguez-Alvarez, Marta
Kim, Daria
Khobta, Andriy
author_facet Rodriguez-Alvarez, Marta
Kim, Daria
Khobta, Andriy
author_sort Rodriguez-Alvarez, Marta
collection PubMed
description 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.
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spelling pubmed-73571512020-07-23 EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions Rodriguez-Alvarez, Marta Kim, Daria Khobta, Andriy Biomolecules Article 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. MDPI 2020-06-13 /pmc/articles/PMC7357151/ /pubmed/32545792 http://dx.doi.org/10.3390/biom10060902 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Rodriguez-Alvarez, Marta
Kim, Daria
Khobta, Andriy
EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions
title EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions
title_full EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions
title_fullStr EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions
title_full_unstemmed EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions
title_short EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions
title_sort egfp reporters for direct and sensitive detection of mutagenic bypass of dna lesions
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357151/
https://www.ncbi.nlm.nih.gov/pubmed/32545792
http://dx.doi.org/10.3390/biom10060902
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