(A) Schematic of alt-EJ reaction and subsequent procedures used for amplification and sequencing of end-joining products. Control alt-EJ reactions were performed with 10 mM Mg2+ and 1 mM Mn2+. (B) Non-denaturing gels showing the products of PCR reactions containing either purified DNA from alt-EJ reactions performed in the presence of Polθ and Lig3 (top left), Polθ alone (top middle), and in the absence of Polθ and Lig3 (top right), or no DNA with primers only (bottom middle). Products in the top middle and top right gels are due to primer-dimer events as shown in the primers only control (bottom middle gel). Lanes 1-8 represent PCR reactions performed at the following respective temperatures: 61°C, 60.8°C, 60.4°C, 59.9°C, 59.2°C, 58.6°C, 58.2°C, 58°C. Lanes 9–13 represent PCR reactions performed in the absence of PCR primers RP435 and RP431 and at the following respective temperatures: 61°C, 60.4°C, 59.9°C, 59.2°C, 58.2°C. The absence of PCR products in lanes 9–13 show that Taq polymerase cannot amplify original pssDNA templates via end-joining or other mechanisms. (C) Plot showing percent of end-joining products observed in cloning vectors following end-joining reactions containing the indicated proteins. Red, end-joining products with insertions; grey, end-joining products without insertions. n = 64 (+Polθ, +Lig3), n = 72 (+Polθ, –Lig3), n =12 (–Polθ, –Lig3). End-joining products in the absence of Polθ and Lig3 are likely due to infrequent byproducts of PCR.