A cell-free system for mitotically-stimulated TMEJ
- 1Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
- 2Howard Hughes Medical Institute
Abstract
Double-stranded DNA breaks (DSBs) are cytotoxic and must be repaired to avoid cell death or mutagenesis. DSBs that form in the S or G2 phases of the cell cycle undergo end resection, creating substrates for homologous recombination (HR) and alternative end joining pathways. Theta-mediated end joining (TMEJ) is a DNA polymerase {theta} (Pol {theta}) dependent repair pathway that joins resected DSB ends by annealing short regions of microhomology. TMEJ becomes particularly important when HR is compromised or during mitosis. Despite intensive investigation, the mechanism of TMEJ remains poorly understood, in part due to the lack of an efficient cell-free system that recapitulates this reaction. Here, we address this deficit using Xenopus egg extracts. We show that a linear DNA template containing a 3' single-stranded DNA overhang with terminal microhomology undergoes efficient intermolecular joining in nucleoplasmic extracts derived from Xenopus eggs. Joining is abolished upon Pol {theta} depletion, and restoration of joining requires Pol {theta} that is proficient in both ATPase and DNA polymerase activities. Furthermore, joining is accelerated by recombinant Cyclin B-CDK1, which also enhances the recruitment of Pol {theta} to single-stranded DNA ends. This assay provides a biochemically tractable approach for dissecting the mechanism and regulation of TMEJ in a physiologically complex environment.
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BibTeX
@misc{wu2026cellfree,
title = {{A cell-free system for mitotically-stimulated TMEJ}},
author = {Yufan Wu and Martha J. Longley and Geylani Can and Johannes C. Walter and Joseph J. Loparo},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.26.762058},
url = {https://www.biorxiv.org/content/10.64898/2026.10.26.762058v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - A cell-free system for mitotically-stimulated TMEJ
AU - Yufan Wu
AU - Martha J. Longley
AU - Geylani Can
AU - Johannes C. Walter
AU - Joseph J. Loparo
PY - 2026
DA - 2026/10/26
PB - bioRxiv
DO - 10.64898/2026.10.26.762058
UR - https://www.biorxiv.org/content/10.64898/2026.10.26.762058v1
AB - Double-stranded DNA breaks (DSBs) are cytotoxic and must be repaired to avoid cell death or mutagenesis. DSBs that form in the S or G2 phases of the cell cycle undergo end resection, creating substrates for homologous recombination (HR) and alternative end joining pathways. Theta-mediated end joining (TMEJ) is a DNA polymerase {theta} (Pol {theta}) dependent repair pathway that joins resected DSB ends by annealing short regions of microhomology. TMEJ becomes particularly important when HR is compromised or during mitosis. Despite intensive investigation, the mechanism of TMEJ remains poorly understood, in part due to the lack of an efficient cell-free system that recapitulates this reaction. Here, we address this deficit using Xenopus egg extracts. We show that a linear DNA template containing a 3' single-stranded DNA overhang with terminal microhomology undergoes efficient intermolecular joining in nucleoplasmic extracts derived from Xenopus eggs. Joining is abolished upon Pol {theta} depletion, and restoration of joining requires Pol {theta} that is proficient in both ATPase and DNA polymerase activities. Furthermore, joining is accelerated by recombinant Cyclin B-CDK1, which also enhances the recruitment of Pol {theta} to single-stranded DNA ends. This assay provides a biochemically tractable approach for dissecting the mechanism and regulation of TMEJ in a physiologically complex environment.
SN - 2692-8205
ER -