Table 4

Experimental assays that are used for the detection of replicative stress

AssayBrief descriptionReferences
DNA fiber assayThis method relies on the sequential incorporation of two thymidine analogs, usually 5-iodo-2’-deoxyuridine (IdU) and 5-chloro-2’-deoxyuridine (CldU), which generates epitopes for fluorescent antibodies. After labeling, cells are fixed and the DNA fibers are spread on glass slides for immunostaining and microscopic visualization. The average length of fluorescent labels on ssDNA enables monitoring of replication fork progression and number of active origins2
RAD51 foci formationRAD51 forms nuclear foci at the sites of DSBs, and this can be visualized by immunofluorescent labeling and microscopic detection63
Nascent iPONDMethod used for the identification of proteins that are recruited at replication forks. This technique requires labeling of newly replicated DNA with modified nucleoside analogs. Fixation of protein-DNA complex (similar to chromatin immunoprecipitation) and immobilization of labeled DNA on beads are analyzed through mass spectrometry or immunoblot analysis2
Neutral comet assayTechnique based on micro-electrophoresis of single cell DNA content to measure the presence of ssDNA breaks. Cells embedded in agar are subjected to migration under alkaline pH. Undamaged DNA migrates slowly giving head of the 'comet' appearance, whereas DNA fragments (from ssDNA breaks) migrate more quickly generating tail of the comet. The amount of DNA tails directly correlates with the percentage of DNA damage2
Patient derived organoidsHill and colleagues developed 33 patient-derived organoid cultures from patients with HGSOC and tested them for HR defects, as well as replication fork protection. Functional deficiencies in fork protection was associated with sensitivity to CHK1i, ATRi, and platinum therapy. This study provides proof-of-concept of a potential model for functional individualized replicative stress testing64
  • CldU, 5-chloro-2’-deoxyuridine; DSBs, double-stranded DNA breaks; HGSOC, high grade serous ovarian cancer; HR, homologous recombination; IdU, 5-iodo-2’-deoxyuridine; iPOND, isolation of proteins on nascent DNA; ssDNA, single-stranded DNA.