Fig. 1: Pol θ can perform end-joining with diverse paired ssDNAs.

a Schematic of Pol θ Δcen. Domains are shown with amino acid boundaries for human Pol θ. The polyacrylamide gel shows purified human Pol θ Δcen, which was similar in at least three different purification runs. b Overall TMEJ process and questions to be answered. Created in BioRender. Li, Y. (2025) https://BioRender.com/vxre3ij. c End-joining of two sets of paired oligonucleotides by Pol θ Δcen. The schematic shows two sets of paired oligonucleotides containing 5ʹ fluorescent labels and 3ʹ terminal or internal 6 bp MH. Reaction mixtures incubated at different time points were separated by electrophoresis on a native 10% polyacrylamide gel (n = 2). Intensities were measured with ImageJ, and the percentage of TMEJ products is labeled under the corresponding lanes. A magenta triangle indicates the starting Cy5-labeled oligonucleotide, and a green star indicates the starting Cy3-labeled oligonucleotide. d Verification of end-joining products from a single experiment. The schematic shows a diagnostic BamHI site and the fragment size of end-joining products after digestion. Created in BioRender. Li, Y. (2025) https://BioRender.com/vxre3ij. The fluorescence colors of the fragments are indicated. Electrophoresis of end-joining products was conducted with or without BamHI digestion. The digested large fragment (∼152 bp) is labeled with an arrow. e Sequence alignment of products after end-joining of Oligo 62 and Oligo 7, which have three unpaired nucleotides adjacent to an 8 bp MH, as shown at the bottom. Source data are provided as a Source Data file.