Pol θ-Mediated End-Joining Uses Microhomologies Containing Mismatches

Authors

Yuzhen Li
Ngoc K Dang
Wei He
Mark Returan
Denisse Carvajal-Maldonado
Adele T Guerin
Han Xu
Bin Liu
Richard D Wood

Publication Date

7-2-2025

Journal

Nature Communications

DOI

10.1038/s41467-025-61258-3

PMID

40603872

PMCID

PMC12222517

PubMedCentral® Posted Date

7-2-2025

PubMedCentral® Full Text Version

Post-print

Abstract

DNA polymerase theta (Pol θ) initiates repair of DNA double-strand breaks by pairing single strands at short "microhomologies". It is important to understand microhomology selection, as some cancer cells rely on Pol θ for survival. Here, we investigate end-joining by purified human Pol θ, employing DNA sequencing of products generated from oligonucleotide libraries having diverse 3' ends. Pol θ overwhelmingly selects short internal microhomologies found within 15 nucleotides of the terminus of single-stranded DNAs, restricting deletion size during end-joining. Significantly, we find that the selected microhomologies are usually interrupted by mismatches and that base pairing within 6 nucleotides of the 3' end is important for determining microhomology choice. Bidirectional synthesis is not necessary to initiate end-joining. The preference for mismatched microhomologies suggests a revision of the definition of microhomology to account for the unique properties of Pol θ. This could advance the analysis of mutations in cancer genomes.

Keywords

Humans, DNA Polymerase theta, DNA-Directed DNA Polymerase, DNA End-Joining Repair, DNA Breaks, Double-Stranded, Base Pair Mismatch, DNA, Single-Stranded, Double-strand DNA breaks, Enzyme mechanisms

Published Open-Access

yes

Recommended Citation

Li, Yuzhen; Dang, Ngoc K; He, Wei; et al., "Pol θ-Mediated End-Joining Uses Microhomologies Containing Mismatches" (2025). Faculty, Staff and Student Publications. 4756.
https://digitalcommons.library.tmc.edu/uthgsbs_docs/4756