Single-mitosis dissection of acute and chronic DNA mutagenesis and repair

Area of Science:

• Genomics
• Cancer Biology
• Molecular Evolution

Background:

• The evolution of the cancer genome is driven by complex mutational processes, including chronic DNA damage and acute bursts of damage, but the precise mechanisms remain unclear.
• Understanding the interplay between different mutational signatures is crucial for developing effective cancer therapies.

Purpose of the Study:

• To develop and validate a strategy for disentangling and quantifying distinct mechanisms of genome evolution at single-cell and single-strand resolution.
• To differentiate between chronic (reactive oxygen species - ROS) and acute (ultraviolet light - UV) mutagenesis in driving cancer genome alterations.

Main Methods:

• Microfluidic separation of sister cells after burst UV damage to analyze mutations at single mitosis.
• Single-strand DNA sequencing to resolve mutation phasing across the genome.
• Analysis of mutation patterns in liver tumors from F1 mice.

Main Results:

• Ultraviolet (UV) mutations were observed as sister-specific events with mirror-image phasing genome-wide.
• Reactive oxygen species (ROS) mutagenesis in transcribed regions showed strand-agnostic reduction.
• Replication over persistent UV damage led to multiallelic variation at CC dinucleotides.
• Mutation phasing was successfully resolved to single strands in mouse liver tumors.

Conclusions:

• The developed strategy effectively distinguishes contributions from overlapping, cancer-relevant mutational processes.
• This approach provides a powerful tool for dissecting the complex landscape of cancer genome evolution.
• Findings offer new insights into how different types of DNA damage shape the cancer genome over time.