Exceptional origin activation revealed by comparative analysis in two laboratory yeast strains

• Published in: PloS one Vol. 17; no. 2; p. e0263569
• Main Authors: Joshi, Ishita, Peng, Jie, Alvino, Gina, Kwan, Elizabeth, Feng, Wenyi
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14-02-2022; Public Library of Science (PLoS)
• Subjects: Activation analysis; Binding sites; Biochemistry; Biology and Life Sciences; Cell cycle; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Checkpoint Kinase 2 - genetics; Checkpoint Kinase 2 - metabolism; Comparative analysis; Conserved sequence; Deoxyribonucleic acid; DNA; DNA replication; DNA, Fungal - genetics; Gene expression; Gene mapping; Genetic aspects; Genetic research; Genomes; Hydroxyurea; Kinases; Laboratories; Molecular biology; Mutation; Nucleotide sequence; Origins; Phosphorylation; Polymorphism; Polymorphism, Single Nucleotide; Reductases; Reduction; Replication fork; Replication Origin; Replication origins; Research and Analysis Methods; S Phase Cell Cycle Checkpoints; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Single-stranded DNA; Transcription; Transcription, Genetic; Whole Genome Sequencing; Yeast; Yeast fungi; Yeasts; United States; New York; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0263569

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 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.