G-quadruplexes (G4) are potential DNA roadblocks that perturb mitochondrial replication machinery and pose a clinically relevant source of mitochondrial DNA mutation and instability

线粒体（mt）基因组的突变与癌症和衰老有关。G-四链体（G4），在富含鸟嘌呤（G）的模板中产生的动态DNA结构，可能使mt复制体停滞，从而促进诱变。我们使用来自两个意大利队列的基因组序列数据的计算分析来证明G4 s和mt变异之间的关联。使用G4 Hunter软件预测mtDNA中G4形成区，我们发现在稳定的G4区中突变显著富集。体外生物化学数据表明，G4有效地阻断mt复制聚合酶γ（Pol）。添加MT复制体相关因子，包括TWINKLE解旋酶和MT单链结合蛋白，不能通过G4阻断刺激pol合成。我们发现，其他mt聚合酶进一步催化易错核苷酸掺入G4结构，表明其参与G4旁路伴随突变风险增加。总之，计算和生物化学方法表明，mt点突变富集在稳定的G4结构，与复制体停滞在G-四链体和依赖于易错的DNA合成一致。

Mutations in the mitochondrial (mt) genome are correlated with cancer and aging. G-quadruplexes (G4), dynamic DNA structures that arise in guanine (G)-rich templates, potentially stall the mt replisome and thereby promote mutagenesis. We used computational analyses of genome sequence data from two Italian cohorts to demonstrate an association between G4s and mt variation. Using the software G4Hunter to predict G4-forming regions in mtDNA, we found statistically significant enrichment of mutations in stable G4 regions. In vitro biochemical data demonstrated that G4s potently block the mt replicative polymerase gamma (Pol ). Addition of mt replisome-associated factors including TWINKLE helicase and mt single-strand binding protein were unable to stimulate pol synthesis through the G4 block. We showed that other mt polymerases further catalyzes error-prone nucleotide incorporation into G4 structures, suggesting its involvement in G4 bypass with accompanying increased risk of mutation. Altogether, the computational and biochemical approaches indicate that mt point mutations are enriched at stable G4 structures, consistent with replisome stalling at G-quadruplexes and reliance on error-prone DNA synthesis.