Advancing inducible gene-inactivation systems to explore synthetic lethality

Area of Science:

• Molecular Biology
• Genetics
• Cancer Research

Background:

• Synthetic lethality is a promising strategy for targeted cancer therapy.
• Current gene knockout and RNAi methods have limitations in studying gene interactions, especially those causing cell death.

Purpose of the Study:

• To develop a novel platform for precise gene inactivation to overcome limitations of existing methods.
• To investigate the functional interplay between BRCA1 and RAD18 using the new platform.

Main Methods:

• Development of the Inducible Gene-Inactivation Systems (IGIS) platform for tetracycline-regulated gene silencing.
• Application of IGIS in human cell lines combined with cell survival, DNA fiber, BrdU alkaline comet assays, and pRPA immunostaining.
• Investigating the synthetic-lethal interaction between BRCA1 and RAD18.

Main Results:

• The IGIS platform allows precise timing of gene inactivation, avoiding variability.
• BRCA1 and RAD18 function in distinct pathways for replication-fork restart and post-replicative gap filling.
• Combined loss of BRCA1 and RAD18 leads to ssDNA gap accumulation and replication catastrophe.

Conclusions:

• The IGIS platform is a valuable tool for studying gene interactions and synthetic lethality.
• RAD18-dependent DNA damage tolerance mechanisms represent potential therapeutic vulnerabilities in BRCA-deficient cancers.