A cell-free system for mitotically-stimulated TMEJ

A Xenopus nucleoplasmic cell-free system shows efficient intermolecular TMEJ of linear DNA with a 3' single-stranded overhang and terminal microhomology.

• Why it matters: DSB repair during S/G2 and mitosis relies on pathways like TMEJ, but its mechanism is poorly understood due to the lack of an efficient cell-free assay.
• What they did: Using Xenopus egg extracts, the authors test joining of a linear DNA template with terminal microhomology, then deplete Pol theta and restore it with ATPase- and polymerase-proficient Pol theta.
• The result: Joining is abolished by Pol theta depletion and accelerated by recombinant Cyclin B-CDK1, which enhances Pol theta recruitment to single-stranded DNA ends, enabling mechanistic dissection in a complex environment.