DNA polymerase theta (Pol θ) is an error-prone translesion polymerase that becomes crucial for DNA double-strand break repair when cells are deficient in homologous recombination or non-homologous end joining. In some organisms, Pol θ also promotes tolerance of DNA interstrand crosslinks. Due to its importance in DNA damage tolerance, Pol θ is an emerging target for treatment of cancer and disease. Prior work has characterized the functions of the Pol θ helicase-like and polymerase domains, but the roles of the linker domain are largely unknown. Here, we show that the Drosophila melanogaster Pol θ linker domain promotes proper egg development and is required for repair of DNA double-strand breaks and interstrand crosslink tolerance. While a linker domain with scrambled amino acid residues is sufficient for DNA repair, replacement of the linker with part of the Homo sapiens Pol θ linker or a disordered region from the FUS RNA-binding protein does not restore function. These results demonstrate that the linker domain is not simply a random tether between the catalytic domains and suggest that intrinsic amino acid residue properties, rather than protein interaction motifs, are more critical for Pol θ linker functions in DNA repair.
© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.