Evaluation of repair activity by quantification of ribonucleotides in the genome,Genes to Cells

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Evaluation of repair activity by quantification of ribonucleotides in the genome
Genes to Cells ( IF 1.3 ) Pub Date : 2021-05-15 , DOI: 10.1111/gtc.12871
Tetsushi Iida ₁ , Naoko Iida _{2,

3} , Jun Sese ₄ , Takehiko Kobayashi _{1,

5}

Affiliation

Ribonucleotides incorporated in the genome are a source of endogenous DNA damage and also serve as signals for repair. Although recent advances of ribonucleotide detection by sequencing, the balance between incorporation and repair of ribonucleotides has not been elucidated. Here, we describe a competitive sequencing method, Ribonucleotide Scanning Quantification sequencing (RiSQ-seq), which enables absolute quantification of misincorporated ribonucleotides throughout the genome by background normalization and standard adjustment within a single sample. RiSQ-seq analysis of cells harboring wild-type DNA polymerases revealed that ribonucleotides were incorporated nonuniformly in the genome with a 3′-shifted distribution and preference for GC sequences. Although ribonucleotide profiles in wild-type and repair-deficient mutant strains showed a similar pattern, direct comparison of distinct ribonucleotide levels in the strains by RiSQ-seq enabled evaluation of ribonucleotide excision repair activity at base resolution and revealed the strand bias of repair. The distinct preferences of ribonucleotide incorporation and repair create vulnerable regions associated with indel hotspots, suggesting that repair at sites of ribonucleotide misincorporation serves to maintain genome integrity and that RiSQ-seq can provide an estimate of indel risk.

中文翻译：

通过量化基因组中的核糖核苷酸来评估修复活性

基因组中掺入的核糖核苷酸是内源性 DNA 损伤的来源，也可作为修复信号。尽管通过测序检测核糖核苷酸的最新进展，但尚未阐明核糖核苷酸掺入和修复之间的平衡。在这里，我们描述了一种竞争性测序方法，即核糖核苷酸扫描定量测序 (RiSQ-seq)，它可以通过背景标准化和单个样本中的标准调整，对整个基因组中错误掺入的核糖核苷酸进行绝对量化。对含有野生型 DNA 聚合酶的细胞进行 RiSQ-seq 分析表明，核糖核苷酸不均匀地掺入基因组中，具有 3' 位移分布和对 GC 序列的偏好。尽管野生型和修复缺陷突变菌株中的核糖核苷酸谱显示出相似的模式，但通过 RiSQ-seq 对菌株中不同核糖核苷酸水平的直接比较能够以碱基分辨率评估核糖核苷酸切除修复活性，并揭示修复的链偏向性。核糖核苷酸掺入和修复的不同偏好创造了与插入缺失热点相关的脆弱区域，这表明核糖核苷酸错误掺入位点的修复有助于保持基因组完整性，并且 RiSQ-seq 可以提供插入缺失风险的估计。

更新日期：2021-05-15

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