Molecular basis of microhomology-mediated end-joining by purified full-length Polθ

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA polymerase θ (Polθ) is a unique fusion protein with polymerase and helicase activities.
• Polθ plays a critical role in microhomology-mediated end-joining (MMEJ), a pathway for repairing DNA double-strand breaks (DSBs).
• The precise molecular mechanism of full-length human Polθ in MMEJ is not fully understood.

Purpose of the Study:

• To elucidate the molecular mechanisms by which full-length human Polθ mediates MMEJ.
• To investigate the roles of the helicase, polymerase-helicase attachment, and central domain in Polθ-dependent MMEJ.
• To understand the structural organization of Polθ during MMEJ.

Main Methods:

• Biochemical assays to study Polθ activity on single-stranded DNA (ssDNA) overhangs, modeling resected DSBs.
• Single-particle microscopy to visualize Polθ complex formation.
• Biophysical methods to characterize Polθ-DNA interactions and complex structures.

Main Results:

• The helicase activity of Polθ is essential for MMEJ of long ssDNA overhangs.
• Polθ MMEJ requires polymerase-helicase attachment but not the helicase's ATPase activity or the disordered central domain.
• Polymerase-helicase attachment promotes the formation of multimeric, gel-like Polθ complexes that facilitate DNA accumulation, synapsis, and MMEJ.
• The central domain regulates Polθ multimerization and influences its DNA substrate specificity for MMEJ.

Conclusions:

• The helicase and polymerase-helicase tethering are crucial for Polθ-mediated MMEJ.
• Polθ forms unique multimeric structures essential for efficient DNA repair.
• The central domain plays a regulatory role in Polθ complex formation and substrate selection during MMEJ.
• These findings reveal novel functions for Polθ domains and highlight the importance of its structural organization in DNA repair.