Evaluation of Repair Activity by Quantification of Ribonucleotides in the Genome

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 non-uniformly 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.整合进基因组的核糖核苷酸是内源性DNA损伤的来源之一，同时也可作为DNA修复的信号分子。尽管当前核糖核苷酸的测序检测技术已取得诸多进展，但基因组中核糖核苷酸的掺入与修复之间的动态平衡仍未得到阐明。本研究报道了一种竞争性测序方法——核糖核苷酸扫描定量测序（Ribonucleotide Scanning Quantification sequencing, RiSQ-seq），该方法可通过单样本内的背景归一化与标准校准，实现全基因组范围内错掺入核糖核苷酸的绝对定量。对携带野生型DNA聚合酶的细胞开展RiSQ-seq分析后发现，基因组内的核糖核苷酸掺入呈现非均匀分布特征，具有3’端偏移的分布模式，且偏好GC序列。尽管野生型菌株与修复缺陷突变菌株中的核糖核苷酸分布模式较为相似，但通过RiSQ-seq直接比较两类菌株间的核糖核苷酸水平，可在碱基分辨率下评估核糖核苷酸切除修复活性，并揭示了修复过程的链偏好性。核糖核苷酸掺入与修复的不同偏好性会形成与插入缺失（indel）热点区域相关的基因组易感位点，这表明核糖核苷酸错掺入位点的修复有助于维持基因组完整性，而RiSQ-seq可用于评估插入缺失风险。