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In response to DNA damage, eukaryotic cells activate a checkpoint signalling cascade, resulting in cell cycle arrest, stabilisation of replication forks and activation of repair. While many players in these pathways have been identified, little is known about the original sensors, or of the DNA structures involved. Because it is present in all checkpoint-inducing lesions, single-stranded DNA (ssDNA) is a good candidate for a common structure recognised by the DNA damage response. The role of ssDNA in checkpoint activation in the yeast Saccharomyces cerevisiae was investigated using three different approaches. Firstly, an attempt was made to produce ssDNA independently of strand breaks by inducing replication-independent plasmid unwinding. Secondly, the effects of depleting the major ssDNA-binding complex, replication protein A (RPA) were analysed. Lastly, an assay to quantify ssDNA generated at a defined DNA double-strand break (DSB) was developed. Despite extensive efforts, the first approach proved unsuccessful, as the method used did not generate unwound plasmid. Using the second approach, it was found that depletion of RPA did not inhibit checkpoint activation during replication stress. Furthermore, replication with limiting amounts of RPA led to rapid cell death and checkpoint activation that was mediated independently of the response to stalled replication forks. Lastly, at a defined DSB it was found that less ssDNA was being generated than had previously been estimated from results based on non-quantitative methods. Additionally, an element of dose dependency was observed in the checkpoint response to DSBs, with stronger and more rapid responses being generated by higher numbers of breaks. Formation of four DSBs resulted in checkpoint activation even in G1 arrested cells. Together, these results raise the possibility of a DNA damage checkpoint pathway largely independent of long tracts of RPA-coated ssDNA and show that checkpoint activation to DSB-damage is possible in G1.