A novel imaging biosensor for the detection of reversed replication forks and four-way junctions in human cells

A groundbreaking technique has been developed to detect reversed replication forks and four-way junctions within human cells using a novel imaging biosensor. Replication fork reversal and recombination-dependent replication create these DNA intermediates, which are crucial for maintaining genomic stability but have been challenging to observe in living cells due to the need for bulk DNA analysis via electron microscopy.

This new biosensor, genetically encoded and targeted to the nucleus, utilizes bacterial RuvA fused with either GFP or Spot-tag. By employing SIRF (Single Molecule Real-Time Fluorescence), the biosensor selectively accumulates at nascent DNA following replication stress induced by hydroxyurea or camptothecin. Its specificity is confirmed by the fact that recruitment of the biosensor requires fork-reversal factors and is inhibited by a non-binding mutant (K84E/K119E).

The biosensor's dynamic tracking of reversed fork abundance allows for the visualization of MRE11-mediated fork degradation in BRCA2- or RAD52-deficient cells, as well as the functional rescue of this process. Additionally, the tool enables the observation of RAD51-dependent four-way junctions at nucleolar rDNA arrays in both normal and stressed cells.

This biosensor provides a visualizable readout with single-cell and subnuclear resolution, allowing for direct spatial analysis of DNA structures within living cells.