Polλ promotes microhomology-mediated end-joining

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Structural Biology

Background:

• Microhomology-mediated end-joining (MMEJ) is a DNA double-strand break (DSB) repair pathway.
• MMEJ is traditionally considered dependent on DNA polymerase theta (Polθ) and independent of nonhomologous end-joining (NHEJ) factors.
• The existence of alternative MMEJ pathways remained an open question.

Purpose of the Study:

• To investigate whether MMEJ is exclusively mediated by Polθ or if other pathways exist.
• To explore the role of DNA polymerase lambda (Polλ) in MMEJ.
• To elucidate the mechanism and structural basis of Polλ-mediated MMEJ.

Main Methods:

• Cellular assays in mammalian cells to assess MMEJ activity.
• Genetic analysis involving NHEJ factors (LIG4/XRCC4) and Polθ.
• X-ray crystallography of Polλ at 2.0 Å resolution using in situ photo-induced DSB formation.

Main Results:

• Human Polλ exhibits robust MMEJ activity, similar to Polθ.
• Polλ promotes MMEJ independently of essential NHEJ factors (LIG4/XRCC4) and Polθ, indicating a distinct Polλ-dependent MMEJ mechanism.
• X-ray crystallography captured Polλ stabilizing a microhomology DNA synapse and performing replication across it.
• Polλ was found to be semisynthetic lethal with BRCA1 and BRCA2.

Conclusions:

• DNA polymerase lambda (Polλ) mediates a distinct MMEJ pathway for DNA double-strand break repair.
• This Polλ-dependent MMEJ mechanism operates independently of canonical NHEJ factors and Polθ.
• The structural insights reveal how Polλ functions in joining DNA ends via microhomology recognition and replication.
• Polλ represents a potential therapeutic target in cancer, given its synthetic lethality with BRCA1/2.