Sequential Requirements for Distinct Polθ Domains During Theta-Mediated End Joining

Authors

Carel Fijen
Lea Drogalis Beckham
Dante Terino
Yuzhen Li
Dale A Ramsden
Richard D Wood
Sylvie Doublié
Eli Rothenberg

Publication Date

4-18-2024

Journal

Molecular Cell

Abstract

DNA polymerase θ (Polθ) plays a central role in a DNA double-strand break repair pathway termed theta-mediated end joining (TMEJ). TMEJ functions by pairing short-sequence "microhomologies" (MHs) in single-stranded DNA at each end of a break and subsequently initiating DNA synthesis. It is not known how the Polθ helicase domain (HD) and polymerase domain (PD) operate to bring together MHs and facilitate repair. To resolve these transient processes in real time, we utilized in vitro single-molecule FRET approaches and biochemical analyses. We find that the Polθ-HD mediates the initial capture of two ssDNA strands, bringing them in close proximity. The Polθ-PD binds and stabilizes pre-annealed MHs to form a synaptic complex (SC) and initiate repair synthesis. Individual synthesis reactions show that Polθ is inherently non-processive, accounting for complex mutational patterns during TMEJ. Binding of Polθ-PD to stem-loop-forming sequences can substantially limit synapsis, depending on the available dNTPs and sequence context.

Keywords

DNA-Directed DNA Polymerase, DNA Breaks, Double-Stranded, DNA Replication, DNA, Single-Stranded, DNA Helicases, DNA End-Joining Repair, DNA polymerase theta, DNA repair, Polθ, TMEJ, double-strand breaks, helicase, microhomology, single-molecule FRET, stem-loops

DOI

10.1016/j.molcel.2024.03.010

PMID

38640894

PMCID

PMC11031631

PubMedCentral® Posted Date

4-18-2025

PubMedCentral® Full Text Version

Author MSS

Published Open-Access

yes