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.
Recommended CitationChandramouly, Gurushankar; Zhao, Jiemin; McDevitt, Shane; Rusanov, Timur; Hoang, Trung; Borisonnik, Nikita; Treddinick, Taylor; Lopezcolorado, Felicia Wednesday; Kent, Tatiana; Siddique, Labiba; Mallon, Joseph; Huhn, Jacklyn; Shoda, Zainab; Kashkina, Ekaterina; Brambati, Alessandra; Stark, Jeremy M; Chen, Xiaojiang S; and Pomerantz, Richard, "Polθ reverse transcribes RNA and promotes RNA-templated DNA repair" (2021). Department of Biochemistry and Molecular Biology Faculty Papers. Paper 186.
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