Sequential requirements for distinct Polθ domains during theta-mediated end joining

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Enzymology

Background:

• DNA polymerase theta (Polθ) is crucial for the theta-mediated end joining (TMEJ) DNA repair pathway.
• TMEJ involves pairing microhomologies (MHs) on single-stranded DNA ends to initiate repair synthesis.
• The precise roles of Polθ's helicase (HD) and polymerase (PD) domains in MH synapsis and repair remain unclear.

Purpose of the Study:

• To elucidate the real-time molecular mechanisms by which Polθ facilitates microhomology-mediated DNA repair.
• To investigate the distinct functions of the Polθ helicase and polymerase domains in the TMEJ pathway.

Main Methods:

• In vitro single-molecule Förster Resonance Energy Transfer (smFRET) assays.
• Biochemical analyses of DNA polymerase activity and protein-DNA interactions.

Main Results:

• The Polθ helicase domain (HD) mediates the initial capture and proximity of two single-stranded DNA (ssDNA) strands.
• The Polθ polymerase domain (PD) stabilizes pre-annealed microhomologies, forming a synaptic complex (SC) to initiate DNA synthesis.
• Polθ exhibits inherent non-processivity, contributing to the observed mutational signatures in TMEJ.
• Polθ-PD binding to specific DNA secondary structures (stem-loops) can impede synapsis, influenced by nucleotide availability and sequence context.

Conclusions:

• The study reveals a sequential mechanism for Polθ function in TMEJ, with HD for DNA capture and PD for stabilization and synthesis.
• Polθ's non-processive nature is a key determinant of TMEJ fidelity and associated mutations.
• Regulatory factors, including DNA sequence and dNTPs, can modulate Polθ's ability to engage in synapsis and repair.