Observing a DNA polymerase choose right from wrong.

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• DNA polymerase β (pol β) is crucial for DNA repair and gap-filling synthesis.
• It utilizes two metal ions for nucleotidyl transfer during catalysis.
• Previous studies relied on substrate analogs to capture catalytic intermediates.

Purpose of the Study:

• To identify novel catalytic intermediates during DNA polymerase β activity.
• To understand the molecular mechanisms underlying polymerase fidelity and genome stability.
• To investigate the role of metal ions in DNA synthesis using natural substrates.

Main Methods:

• Employed natural substrates (correct and incorrect nucleotides) for DNA polymerase β reactions.
• Determined 15 distinct crystal structures to capture real-time product formation.
• Analyzed structural changes at the active site during nucleotide insertion.

Main Results:

• Observed dynamic active site adjustments that favor correct nucleotide insertion and disfavor incorrect insertion.
• Identified a transient third metal binding site formed only during correct nucleotide incorporation.
• Found that pyrophosphate dissociation is faster after incorrect nucleotide insertion, linked to subdomain repositioning.

Conclusions:

• The study reveals previously unappreciated molecular adjustments in DNA polymerase β catalysis.
• A transient third metal site and subdomain movements contribute to polymerase fidelity.
• These findings offer insights into maintaining genome stability through accurate DNA synthesis.