Consistent with this, human recombinant Polθ purified from S. coli and Saccharomyces cerevisiae, respectively) and by different methods ( 1, 9), the observed RT activity is not due to a protein contaminant. Because recombinant human Polθ and Fl-Polθ were purified from different organisms ( E. 1A) ( 1) also has RT activity, suggesting that the endogenous protein performs RNA-DNA repair in cells ( Fig. Full-length Polθ (Fl-Polθ) containing an N-terminal superfamily 2 and disordered central domain ( Fig. All Pols are active on DNA as expected ( Fig. Replicative Pols δ and ε degrade the DNA primer on RNA due to exonuclease activity (fig. Y-family polymerase κ (Polκ) exhibits limited RT activity under various conditions similar to Polη (Fig. Eight other human Pols, representative of at least two enzymes from each polymerase family in humans (A, B, X, and Y), fail to reverse transcribe DNA beyond 2 to 3 nt under conditions identical to those of Polθ ( Fig. The efficient RT activity of Polθ appears to be unique among human Pols. S2B), and Polθ also promotes cDNA synthesis of purified Escherichia coli 16 S ribosomal RNA similar to M-MuLV and AMV RTs (fig. Polθ cDNA synthesis on synthetic RNA is similar to M-MuLV and AMV RTs ( Fig. Polθ exhibits pausing events nearly identical to those of retrovirus RTs, which is consistent with pausing tendencies for RTs ( Fig. In addition to HIV RT, Polθ activity on RNA is nearly identical to RTs encoded by Moloney murine leukemia virus (M-MuLV) and avian myeloblastosis virus (AMV) ( Fig. HIV RT and other retrovirus RTs are also highly error-prone, demonstrating a shared characteristic between Polθ and retroviral RTs ( 18, 24– 28). S2A) and reveals nucleotide misincorporations and indels, which is consistent with its low-fidelity DNA synthesis activity (fig. Complementary DNA (cDNA) sequencing confirms Polθ’s RNA-dependent DNA synthesis activity (fig. Despite its robust activity on RNA, Polθ strongly discriminates against incorporating ribonucleotides (fig. Polθ RNA-dependent DNA synthesis activity is observed under various conditions and on different template constructs (figs. Overall, Polη exhibits increased stalling on RNA and requires higher enzyme concentration relative to Polθ for reverse transcription (figs. Controls show that Polη is active on a DNA/DNA template like Polθ and HIV RT (compare Fig. However, at significantly higher concentrations, Polη can further extend the DNA/RNA (figs. We find that Polη fails to perform reverse transcription beyond 3 nucleotides (nt) using conditions identical to those of Polθ and HIV RT at multiple concentrations ( Fig. Previous studies indicated that human Polη has RT activity at high micromolar concentrations ( 23). Polθ exhibits a similar rate of RT activity as HIV RT under identical conditions using substoichiometric amounts of enzyme relative to template ( Fig. We tested whether the polymerase domain of Polθ (herein referred to as Polθ) reverse transcribes RNA like HIV RT using a DNA primer annealed to a RNA template (DNA/RNA). RESULTS Polθ exhibits RNA-dependent DNA synthesis activity Although RNA-DNA repair mechanisms have been demonstrated in genetically engineered yeast cells ( 21, 22), they remain obscure in mammalian cells. Given that ribonucleotides are the most frequently occurring nucleotide lesion in genomic DNA that arrest replicative Pols and cause DNA breaks ( 19, 20), we also envisaged that Polθ would tolerate template ribonucleotides during its DNA repair activities and thus promote RNA-templated DNA repair synthesis (RNA-DNA repair). Because Polθ is highly error-prone and promiscuous and contains an inactive proofreading domain, we hypothesized that it has RNA-dependent DNA synthesis activity. Inactivating the 3′-5′ proofreading function of closely related A-family bacterial Pol I Klenow fragment (KF) enables this polymerase to reverse transcribe RNA like retroviral reverse transcriptases (RTs), which lack proofreading activity (fig. Intriguingly, Polθ has an inactive proofreading domain due to acquired mutations ( Fig.
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