Structural basis of DNA polymerase theta mediated DNA end joining

DNA polymerase theta (Pol theta) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol theta recognizes microhomologous DNA ends and performs low-fidelity DNA synthesis remain unclear. Here, we present cryo-electron microscope structures of the polymerase domain of Lates calcarifer Pol theta with long and short duplex DNA at up to 2.4 angstrom resolution. Interestingly, Pol theta binds to long and short DNA substrates similarly, with extensive interactions around the active site. Moreover, Pol theta shares a similar active site as high-fidelity A-family polymerases with its finger domain well-closed but differs in having hydrophilic residues surrounding the nascent base pair. Computational simulations and mutagenesis studies suggest that the unique insertion loops of Pol theta help to stabilize short DNA binding and assemble the active site for MMEJ repair. Taken together, our results illustrate the structural basis of Pol theta-mediated MMEJ.