Fig. 1.

Schematic of the DNA fibre-spreading assay. To assess replication fork protection, cells are sequentially incubated with the thymidine analogues CldU and IdU, followed by the addition of a replication stress-inducing agent, most commonly HU. After spreading, fixing and staining, the fibres can be visualised. A second label tract shorter than the first label may be due to nucleolytic degradation of the nascent strand at stalled replication forks, indicating defective fork protection. This protocol can be modified to assay for single-stranded DNA (ssDNA) gaps, by introducing the S1 nuclease which cleaves opposite gaps to result in a shorter second label.