Accelerates Discovery of Translesion Synthesis Inhibitors to Overcome Cancer Chemoresistance

Background

Human cells constantly rely on specialized DNA damage tolerance pathways, particularly Translesion Synthesis (TLS) and single-stranded DNA (ssDNA) gap repair, to maintain genomic stability when replication machinery encounters DNA lesions. While essential for normal cellular survival, cancer cells frequently exploit these bypass mechanisms to survive DNA-damaging chemotherapies, driving treatment resistance and mutagenesis. Consequently, there is a critical need in oncology and molecular biology for robust analytical tools capable of accurately measuring cellular TLS and ssDNA gap repair capacities to elucidate mutagenic pathways and identify novel therapeutic inhibitors.

Despite this need, existing methodologies for evaluating DNA repair capacity suffer from significant technical limitations. Current techniques, such as the S1 nuclease DNA fiber assay and fluorescence-based host cell reactivation (FM-HCR) assays, lack precise control over the exact chemical and structural context of specific DNA lesions within ssDNA gaps. Furthermore, these conventional approaches focus broadly on general DNA repair rather than specifically tracking lesion bypass, and they fail to provide a high dynamic range for quantification. Critically, existing assays cannot determine the fidelity, mutational signatures, or exact nucleotide insertion preferences of repair polymerases at single-nucleotide resolution, limiting their utility for high-throughput pharmaceutical screening.

Technology

This technology is an integrated plasmid-based reporter assay and high-throughput sequencing platform designed to quantitatively analyze Translesion Synthesis (TLS) and single-stranded DNA (ssDNA) gap repair capacity in human cells. The core architecture utilizes a one-pot enzymatic assembly to engineer a defined 15-nucleotide ssDNA gap—incorporating either unmodified controls or site-specific DNA lesions like native or synthetic abasic sites—into the transcribed strand of a NanoLuciferase gene. Following cellular delivery, successful intracellular repair across the gap restores NanoLuciferase expression, which is measured via a dual-luciferase luminescence readout normalized against a co-transfected Firefly luciferase control. The platform also integrates an Amplicon-based TLS-Sequencing (ATLS-Seq) workflow that extracts repaired RNA transcripts, removes carryover plasmid DNA, and leverages Illumina or Oxford Nanopore sequencing to evaluate polymerase fidelity and mutational signatures.

This solution is highly differentiated from existing DNA repair methods, such as S1 nuclease DNA fiber assays and fluorescence-based host cell reactivation, by combining precise lesion control with high-dynamic-range luminescent quantification and single-nucleotide sequencing resolution. Unlike fluorescence platforms that assess general DNA repair without tracking lesion bypass, this technology specifically isolates ssDNA gap bypass and TLS mechanisms. Its ATLS-Seq component uncovers cell-type-specific nucleotide insertion hierarchies and identifies novel repair pathways, such as POLQ-independent Microhomology-Mediated Lesion Skipping. Furthermore, compatibility with genetic perturbations makes it a powerful, high-throughput screening tool for discovering small-molecule TLS inhibitors to combat chemotherapy resistance.

Benefits

• Enables high-throughput quantification of translesion synthesis and single-stranded DNA gap repair capacity using a high-dynamic-range dual-luciferase luminescence readout
• Provides precise control over DNA lesion type and sequence context via a modular, one-pot enzymatic reporter assembly
• Delivers single-nucleotide resolution of repair fidelity, mutational signatures, and polymerase insertion preferences through an integrated amplicon sequencing workflow
• Compatible with both short-read and long-read next-generation sequencing platforms to yield concordant mutational profiles
• Eliminates background interference from unrepaired input plasmids through targeted enzymatic digestion and RNA-specific cDNA amplification
• Facilitates genetic screening and functional characterization of specific DNA repair pathways, accessory factors, and alternative repair mechanisms
• Accelerates pharmaceutical drug discovery by serving as a scalable screening tool to identify small-molecule inhibitors that overcome chemotherapy resistance in cancer cells

Applications

• TLS inhibitor drug screening
• Chemotherapy resistance biomarker profiling
• DNA repair assay kits
• Genotoxicity and mutagenesis profiling
• Synthetic lethality target validation

Additional info

The Laverty lab uses a combination of nucleic acid chemistry, biochemistry, and molecular biology to understand the mechanisms of DNA repair in human cells.