Department of Obstetrics and Gynecology, Ulm University, Ulm 89075, Germany.
Project group Biochemistry, Leibniz Institute on Aging - Fritz Lipmann Institute, D-07745 Jena, Germany.
Nucleic Acids Res. 2020 Dec 2;48(21):12188-12203. doi: 10.1093/nar/gkaa974.
We have previously reported that p53 decelerates nascent DNA elongation in complex with the translesion synthesis (TLS) polymerase ι (POLι) which triggers a homology-directed DNA damage tolerance (DDT) pathway to bypass obstacles during DNA replication. Here, we demonstrate that this DDT pathway relies on multiple p53 activities, which can be disrupted by TP53 mutations including those frequently found in cancer tissues. We show that the p53-mediated DDT pathway depends on its oligomerization domain (OD), while its regulatory C-terminus is not involved. Mutation of residues S315 and D48/D49, which abrogate p53 interactions with the DNA repair and replication proteins topoisomerase I and RPA, respectively, and residues L22/W23, which disrupt formation of p53-POLι complexes, all prevent this DDT pathway. Our results demonstrate that the p53-mediated DDT requires the formation of a DNA binding-proficient p53 tetramer, recruitment of such tetramer to RPA-coated forks and p53 complex formation with POLι. Importantly, our mutational analysis demonstrates that transcriptional transactivation is dispensable for the POLι-mediated DDT pathway, which we show protects against DNA replication damage from endogenous and exogenous sources.
我们之前曾报道过，p53 与跨损伤合成（TLS）聚合酶 ι（POLι）复合物可减缓新生 DNA 延伸，从而触发同源定向 DNA 损伤容忍（DDT）途径，以在 DNA 复制过程中绕过障碍。在这里，我们证明这种 DDT 途径依赖于 p53 的多种活性，这些活性可被包括在肿瘤组织中经常发现的 TP53 突变所破坏。我们表明，p53 介导的 DDT 途径取决于其寡聚结构域（OD），而其调节 C 末端不参与。突变 S315 和 D48/D49 残基可分别使 p53 与 DNA 修复和复制蛋白拓扑异构酶 I 和 RPA 的相互作用，以及 L22/W23 残基破坏 p53-POLι 复合物的形成，均可阻止该 DDT 途径。我们的结果表明，p53 介导的 DDT 需要形成具有 DNA 结合能力的 p53 四聚体，将这种四聚体募集到 RPA 包裹的叉上，并与 POLι 形成 p53 复合物。重要的是，我们的突变分析表明，转录反式激活对于 POLι 介导的 DDT 途径是可有可无的，我们表明该途径可防止内源性和外源性 DNA 复制损伤。
