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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA damage tolerance is crucial for cell survival under genotoxic stress.
• However, this tolerance can lead to errors in DNA replication and transcription.
• Translesion DNA synthesis (TLS) and transcriptional mutagenesis (TM) are key error-prone processes contributing to mutations and potentially cancer.

Purpose of the Study:

• To create reporter systems for detecting DNA modification miscoding during translesion DNA or RNA synthesis in human cells.
• To systematically screen mutations for their impact on reporter function.
• To assess the mutagenic potential of abasic DNA lesions in this system.

Main Methods:

• Systematic phenotypic screening of 25 non-synonymous base substitutions in an enhanced green fluorescent protein (EGFP) DNA sequence.
• Identification of loss-of-fluorescence mutants.
• Incorporation of abasic DNA lesions at mutation sites.

Main Results:

• Identified four loss-of-fluorescence EGFP mutants.
• Demonstrated that subsequent base substitutions at mutated sites can restore EGFP function.
• Showcased high sensitivity in detecting mutagenic DNA TLS and TM using abasic lesions.

Conclusions:

• The developed reporter system effectively detects miscoding during TLS and TM.
• This system provides a sensitive method for studying the mutagenic consequences of DNA damage.
• Findings contribute to understanding cancer development and other diseases linked to transcriptional mutagenesis.