Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template ribonucleotides and promote RNA-templated DNA repair synthesis remains unknown. We find that human Polθ reverse transcribes RNA, similar to retroviral reverse transcriptases (RTs). Polθ exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA. The 3.2-Å crystal structure of Polθ on a DNA/RNA primer-template with bound deoxyribonucleotide reveals that the enzyme undergoes a major structural transformation within the thumb subdomain to accommodate A-form DNA/RNA and forms multiple hydrogen bonds with template ribose 2′-hydroxyl groups like retroviral RTs. Last, we find that Polθ promotes RNA-templated DNA repair in mammalian cells. These findings suggest that Polθ was selected to accommodate template ribonucleotides during DNA repair.

嵌入基因组的核糖核苷酸会阻止复制性 DNA 聚合酶 (Pols) 并导致 DNA 断裂。哺乳动物 DNA 修复 Pols 是否有效地使用模板核糖核苷酸并促进 RNA 模板 DNA 修复合成仍然未知。我们发现人类 Polθ 逆转录 RNA，类似于逆转录病毒逆转录酶 (RT)。与 DNA 相比，Polθ 在 RNA 上表现出更高的脱氧核糖核苷酸掺入速度和保真度。Polθ 在具有结合脱氧核糖核苷酸的 DNA/RNA 引物模板上的 3.2-Å 晶体结构表明，该酶在拇指子域内经历了主要的结构转变以适应 A 型 DNA/RNA 并与模板核糖 2' 形成多个氢键-羟基，如逆转录病毒 RT。最后，我们发现 Polθ 促进哺乳动物细胞中以 RNA 为模板的 DNA 修复。