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

DNA polymerase theta (Pol theta) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Pol theta performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Pol theta MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Pol theta 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 Pol theta complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Pol theta 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 Pol theta.