Global landscape of replicative DNA polymerase usage in the human genome

The division of labour between DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequence (Pu-seq) in HCT116 cells and mapped Pole and Pola usage genome wide. The polymerase usage profiles show Pole synthesises the leading strand and Pola contributes mainly to lagging strand synthesis. Combining the Pole and Pola profiles, we accurately predict the genome-wide pattern of fork directionality, plus zones of replication initiation and termination. We confirm that transcriptional activity contributes toshapes the patterns of initiation and termination and, by separately analysing the effect of transcription ofon both both co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands in both co-directional and convergent forks is compromised by transcription. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability. Overall design: We mapped ribonucleotide-incorporation by the mutated DNA polymerase a and e (POLE1-M630F and POLA1-Y865F) at single-nucleotide resolution in HCT116 cells and subsequent informatics analysis was performed to generate datasets listed here.

DNA聚合酶的分工模式是DNA复制保真性与复制效率的核心基础。然而，人类细胞中复制型DNA聚合酶的具体功能尚未得到直接证实。我们在HCT116细胞中开发了聚合酶使用测序（polymerase usage sequence, Pu-seq）技术，并在全基因组范围内绘制了Pole与Pola的使用图谱。聚合酶使用图谱显示，Pole负责前导链的合成，而Pola主要参与后随链的合成。结合Pole与Pola的使用图谱，我们可精准预测全基因组范围内的复制叉方向性模式，以及复制起始与终止区域。我们证实，转录活性可影响并塑造复制起始与终止的模式；通过分别分析转录对同向及相向复制叉的影响，我们证明，在同向与相向复制叉中，前导链与后随链的偶联DNA合成均会受到转录的干扰。聚合酶解偶联现象在大基因附近尤为显著，包括两个最不稳定的常见脆性位点FRA3B与FRA3D，由此将转录诱导的聚合酶解偶联与染色体不稳定性联系起来。实验总体设计：我们在HCT116细胞中以单碱基分辨率定位了突变型DNA聚合酶α与ε（POLE1-M630F与POLA1-Y865F）的核糖核苷酸掺入情况，并通过后续生物信息学分析生成了本文所列的数据集。