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The Human RAD5 Homologs, HLTF and SHPRH, Have Separate Functions in DNA Damage Tolerance Dependent on the DNA Lesion Type
Open AccessArticle

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

1
Unit “Responses to DNA Lesions", Institute of Toxicology, University Medical Center of the Johannes Gutenberg University Mainz, Obere Zahlbacher Str. 67, 55131 Mainz, Germany
2
Novosibirsk State University, 1 Pirogova St., 630090 Novosibirsk, Russia
3
Laboratory of Genome and Protein Engineering, SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., 630090 Novosibirsk, Russia
*
Author to whom correspondence should be addressed.
Biomolecules 2020, 10(6), 902; https://doi.org/10.3390/biom10060902
Received: 5 May 2020 / Revised: 5 June 2020 / Accepted: 10 June 2020 / Published: 13 June 2020
(This article belongs to the Special Issue DNA Damage Response)
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. View Full-Text
Keywords: DNA damage; DNA damage tolerance; damage bypass; translesion synthesis (TLS); transcriptional mutagenesis; mutation assay; reporter assay; host cell reactivation (HCR); enhanced green fluorescent protein (EGFP) DNA damage; DNA damage tolerance; damage bypass; translesion synthesis (TLS); transcriptional mutagenesis; mutation assay; reporter assay; host cell reactivation (HCR); enhanced green fluorescent protein (EGFP)
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Rodriguez-Alvarez, M.; Kim, D.; Khobta, A. EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions. Biomolecules 2020, 10, 902.

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