Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York, United States of America.
Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.
PLoS One. 2022 Feb 14;17(2):e0263569. doi: 10.1371/journal.pone.0263569. eCollection 2022.
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.
我们通过在受羟基脲（一种核糖核苷酸还原酶抑制剂）挑战的两种酵母菌株中进行全基因组单链 DNA 作图，对复制原点的激活进行了比较分析。我们了解了三个因素对原点激活的影响：S 期检查点控制、DNA 序列多态性和原点与转录单元的相对位置。与 A364a 相比，野生型 W303 的叉进展明显减少，同时 Rad53 磷酸化水平升高，以及原点处的物理存在。此外，与 A364a 相比，W303 中的 rad53K227A 突变体激活了更多的原点，同时导致所有原点的 ssDNA 全面减少。原点的共识基序中的序列多态性在决定菌株特异性活性方面作用较小。最后，我们确定了一类仅在检查点功能正常的细胞中活跃的新原点类，我们将其命名为“Rad53 依赖性原点”。我们的研究提供了一份差异使用原点的综合清单，并为原点激活机制提供了新的见解。
