We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two yeast strains challenged by hydroxyurea, an inhibitor of the ribonucleotide reductase. We gained understanding of the impact on origin activation by three factors: S-phase checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Wild type W303 showed a significant reduction of fork progression accompanied by an elevated level of

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... Rad53 phosphorylation as well as physical presence at origins compared to A364a. Moreover, a rad53K227A mutant in W303 activated more origins, accompanied by global reduction of ssDNA across all origins, compared to A364a. Sequence polymorphism in the consensus motifs of origins plays a minor role in determining strain-specific activity. Finally, we identified a new class of origins only active in checkpoint-proficient cells, which we named "Rad53-dependent origins". Our study presents a comprehensive list of differentially used origins and provide new insights into the mechanisms of origin activation.