Novel insights about ribosomal DNA double strand break repair revealed by super-resolution imaging techniques and very fast CRISPR.

Abstract

Ribosomal DNA (rDNA) is repetitive gene sequence that produces the RNA components for the translation machinery in eukaryotic cells - ribosomes. The maintenance of rDNA genome stability against double-strand breaks (DSBs) is critical regarding cellular aging related diseases and tumor development due to its repetitive nature and distribution over multiple chromosomes. Previous studies have shown that DSB within rDNA region will induce rDNA transcriptional repression and nucleolar segregation. However, it remains poorly understood regarding the kinetics of associated transcriptional regulation, the architecture of the nucleolar cap, and the link between them, likely due to the resolution limit of conventional imaging tools and the absence of proper system that can rapidly induce DSBs specific to rDNA region with accurately controlled timing. To resolve these limitations and provide deeper insights on how rDNA DSB repair response is regulated, we use self-built super-resolution fluorescence imaging systems, e.g., stimulated emission depletion microscopy, which allow to visualize nucleolar structures beyond the diffraction limit, and lattice light sheet microscopy, which enables to monitor fast kinetics of the repair process with minimal photo-induced disturbance, combined with a recently developed rapidly-inducible photo-caged CRISPR-Cas9 system (very fast CRISPR, vfCRISPR) in our lab, to study the spatiotemporal coordination of the DSB repair factor assembly upon rDNA DSB damage, and dissect its important role in regulating pathway choice and outcome fidelity.