Faculty, Staff and Student Publications
Language
English
Publication Date
11-25-2025
Journal
Proceedings of the National Academy of Sciences of the United States of America
DOI
10.1073/pnas.2513018122
PMID
41259142
PMCID
PMC12663940
PubMedCentral® Posted Date
11-19-2025
PubMedCentral® Full Text Version
Post-print
Abstract
DNA polymerase theta (Pol θ)-mediated end joining (TMEJ) initiates DNA double-strand break repair by using short homologies (microhomologies) between single-stranded DNA tails. This repair process is particularly important in cancer cells defective in homologous recombination. The exonuclease function of DNA polymerase delta (Pol δ) has been identified as an essential component for TMEJ, functioning to remove unpaired bases flanking a microhomology (MH). It is not known if the exonuclease removes all unpaired bases at once and how this removal might affect subsequent MH selection. Here, we reconstituted a functional TMEJ repair process using purified human Pol θ and Pol δ. We find that when Pol δ exonuclease excises a nucleotide to generate a new 3' terminus, Pol θ initiates a new MH search. Pol δ exonuclease removes a single nucleotide at a time, rather than cleaving an unpaired flap, and then transfers the DNA tail to the polymerase site of Pol θ. Specific primer-grasp amino acids in the polymerase domain of Pol θ are important for internal MH anchoring. The helicase-like domain of Pol θ harbors two activities which are recapitulated in reconstituted reactions: strand-capture to bring single-strand tails together and an ATPase activity that alleviates suppression of TMEJ by ssDNA binding protein RPA. This functional reconstitution of TMEJ advances the understanding of how two polymerases with three enzymatic activities orchestrate double-strand break repair.
Keywords
Humans, DNA Breaks, Double-Stranded, DNA Polymerase theta, DNA-Directed DNA Polymerase, DNA Polymerase III, DNA End-Joining Repair, DNA Repair, DNA, DNA, Single-Stranded, DNA polymerase, helicase, biochemical reconstitution, TMEJ
Published Open-Access
yes
Recommended Citation
Li, Yuzhen; Returan, Mark; Guerin, Adele T; et al., "Coordinated Transfer of DNA Between Pol θ and Pol δ Resets Microhomology Choice During Double-Strand Break Repair" (2025). Faculty, Staff and Student Publications. 6798.
https://digitalcommons.library.tmc.edu/uthgsbs_docs/6798
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