Department of Microbiology and Molecular Genetics, University of California Davis, One Shields Avenue, Davis, CA 95616, USA.
School of Mathematics and Science, Solano Community College, 4000 Suisun Valley Road, Fairfield, CA 94534, USA.
Nucleic Acids Res. 2022 Feb 28;50(4):2051-2073. doi: 10.1093/nar/gkac035.
Conflicts between transcription and replication machinery are a potent source of replication stress and genome instability; however, no technique currently exists to identify endogenous genomic locations prone to transcription-replication interactions. Here, we report a novel method to identify genomic loci prone to transcription-replication interactions termed transcription-replication immunoprecipitation on nascent DNA sequencing, TRIPn-Seq. TRIPn-Seq employs the sequential immunoprecipitation of RNA polymerase 2 phosphorylated at serine 5 (RNAP2s5) followed by enrichment of nascent DNA previously labeled with bromodeoxyuridine. Using TRIPn-Seq, we mapped 1009 unique transcription-replication interactions (TRIs) in mouse primary B cells characterized by a bimodal pattern of RNAP2s5, bidirectional transcription, an enrichment of RNA:DNA hybrids, and a high probability of forming G-quadruplexes. TRIs are highly enriched at transcription start sites and map to early replicating regions. TRIs exhibit enhanced Replication Protein A association and TRI-associated genes exhibit higher replication fork termination than control transcription start sites, two marks of replication stress. TRIs colocalize with double-strand DNA breaks, are enriched for deletions, and accumulate mutations in tumors. We propose that replication stress at TRIs induces mutations potentially contributing to age-related disease, as well as tumor formation and development.
转录和复制机制之间的冲突是复制应激和基因组不稳定性的一个强大来源；然而，目前还没有技术可以识别容易发生转录-复制相互作用的内源性基因组位置。在这里，我们报告了一种识别容易发生转录-复制相互作用的基因组位点的新方法，称为转录-复制免疫沉淀结合新生 DNA 测序（Transcription- Replication Immunoprecipitation on nascent DNA sequencing，TRIPn-Seq）。TRIPn-Seq 采用顺序免疫沉淀技术，依次沉淀磷酸化的 RNA 聚合酶 2 第 5 位丝氨酸（RNAP2s5），然后富集之前用溴脱氧尿苷标记的新生 DNA。使用 TRIPn-Seq，我们在小鼠原代 B 细胞中绘制了 1009 个独特的转录-复制相互作用（TRIs），其特征是 RNAP2s5 呈双峰模式、双向转录、RNA:DNA 杂交体富集以及形成 G-四链体的概率高。TRIs 在转录起始位点高度富集，并映射到早期复制区域。TRIs 表现出与复制蛋白 A 的增强关联，并且与控制转录起始位点相比，TRI 相关基因的复制叉终止更高，这是复制应激的两个标志。TRIs 与双链 DNA 断裂共定位，富含缺失，并在肿瘤中积累突变。我们提出，TRIs 处的复制应激会导致突变，这些突变可能导致与年龄相关的疾病以及肿瘤的形成和发展。
