Polθ promotes the repair of 5\u27-DNA-protein crosslinks by microhomology-mediated end-joining

DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5\u27 terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5\u27 terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide

Discovery of a Small-Molecule Inhibitor that Traps Polθ on DNA and Synergizes with PARP Inhibitors

The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4-6 nM IC50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi

Molecular basis of microhomology-mediated end-joining by purified full-length Polθ

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ

Polθ reverse transcribes RNA and promotes RNA-templated DNA repair

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\u27-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

Discovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors

Abstract The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4–6 nM IC50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi

Publisher Correction: Molecular basis of microhomology-mediated end-joining by purified full-length Polθ (Nature Communications, (2019), 10, 1, (4423), 10.1038/s41467-019-12272-9)

The original version of this Article contained errors in Figure 6. In panel o, the labels incorrectly stated ‘Poleθ’ and “Poleθ + DNA” and should be labelled “Polθ” and “Polθ + DNA”. In the result section, in the sub-section entitled “Polθ Promotes MMEJ of Long ssDNA”, the sentence “Importantly, the ability of Polθ- pol to perform MMEJ on short (≤12 nt) ssDNA (Fig. 1p, left; Supplementary Fig. 3D and 3E), and short (≤15 nt) overhangs, demonstrates it performs interstrand pairing without Polθ-hel”. should read as follow: “Importantly, the ability of Polθ-pol to perform MMEJ on short (≤12 nt) ssDNA (Fig. 1p, left; Supplementary Fig. 3D and 3E), and short (≤15 nt) overhangs, demonstrates that it performs interstrand pairing without Polθ-hel”. In the sub-section entitled “Preventing Intrastrand Pairing Stimulates MMEJ by Polθ-Pol”, the sentence “We predicted that preventing base-pairing opportunities between 3' terminal bases and bases upstream along long the 5' region of long ssDNA substrates would suppress intrastrand pairing and enable interstrand pairing by Polθ-pol (Fig. 3c)”. should read as follows: “We predicted that preventing base-pairing opportunities between 3' terminal bases and bases upstream along the 5' region of long ssDNA substrates would suppress intrastrand pairing and enable interstrand pairing by Polθ-pol (Fig. 3c)”. In the method section, in the “Proteins” sub-section the sentence “Polθ-pol, Polθ-hel and RPA were purified as described”. should read as follows: “Polθ-pol and Polθ-hel were purified as described”. These corrections have now been included in the HTML and pdf of the article. Additionally, a technical problem during the publication process resulted in loss of image quality in Figs. 1, 3 and 4. This has now been corrected in both the PDF and HTML versions of the Article