Structural basis of error-prone DNA synthesis by DNA polymerase θ

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA polymerase theta (Pol θ) is crucial for DNA double-strand break repair and translesion synthesis.
• Unlike high-fidelity polymerases, Pol θ exhibits a propensity for incorporating errors during DNA synthesis.

Purpose of the Study:

• To elucidate the structural basis for Pol θ's low-fidelity DNA synthesis.
• To understand how Pol θ accommodates and extends from mismatched base pairs.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine structures of Pol θ with various base pairs.
• In-solution fluorescence measurements.
• Mutagenesis studies to identify key residues.

Main Results:

• Pol θ's active site snugly accommodates mismatched base pairs (T:G, T:T) with a closed finger domain.
• Unique active site residues stabilize these mismatches.
• Pol θ efficiently extends from mismatches, leading to insertions and deletions via template or primer looping.

Conclusions:

• Structural insights reveal Pol θ's adaptation for error-prone DNA synthesis.
• The enzyme's unique active site architecture facilitates the stabilization and extension of misincorporated bases.
• These findings explain Pol θ's role in generating genetic diversity through insertions and deletions.