Structural and functional insight into mismatch extension by human DNA polymerase α

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Human DNA polymerase α (Polα) is essential for DNA replication and mutagenesis.
• Polα extends RNA primers, facilitating the initiation of DNA synthesis.
• Unlike other polymerases, Polα lacks proofreading activity.

Purpose of the Study:

• To structurally and functionally analyze human Polα's interaction with mismatched DNA templates.
• To understand how Polα handles primer termini with mismatches during DNA extension.

Main Methods:

• X-ray crystallography to determine the structure of Polα catalytic domain complexed with a mismatched template:primer and dCTP.
• Binding studies at physiological salt concentrations to assess Polα's DNA affinity.
• Pre-steady-state kinetic studies to evaluate primer extension efficiency.

Main Results:

• The crystal structure revealed minimal active site distortion but a planar geometry at the T-C mismatch site.
• Polα exhibits low affinity for DNA and poor discrimination against mismatches without deoxynucleotide triphosphate (dNTP).
• In the presence of cognate dNTP, Polα shows >10-fold selectivity for correct duplexes but extends T-C mismatches 249-fold less efficiently.

Conclusions:

• Human Polα's primer extension is significantly impaired by post-insertion mismatches.
• Mismatch incorporation affects substrate affinity and DNA polymerization rates.
• Understanding Polα's fidelity is crucial for genome stability and preventing mutagenesis.