Crystal structure of Deep Vent DNA polymerase

Area of Science:

• Biochemistry and Molecular Biology
• Structural Biology
• Synthetic Biology

Background:

• DNA polymerases are crucial for DNA replication and biotechnological applications.
• Unnatural base pairs (UBPs) expand the genetic alphabet but require compatible polymerases for replication.
• Deep Vent DNA polymerase shows high fidelity for the Ds-Px UBP, while Taq DNA polymerase exhibits lower performance.

Purpose of the Study:

• To elucidate the structural basis for the differential efficiency and fidelity of DNA polymerases with unnatural base pairs.
• To understand the molecular interactions governing the replication of the 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) unnatural base pair.

Main Methods:

• Determined the crystal structure of Deep Vent DNA polymerase in its apo form at 2.5 Å resolution.
• Constructed structural models of Deep Vent DNA polymerase complexes with DNA containing natural and unnatural bases.
• Analyzed molecular interactions at the active site, focusing on the template-strand DNA and polymerase residues.

Main Results:

• The crystal structure of Deep Vent DNA polymerase was obtained, providing a high-resolution template for modeling.
• Structural models revealed that the unnatural Ds base causes a steric clash with Thr664 in Taq DNA polymerase.
• This clash is absent in Deep Vent DNA polymerase, suggesting a mechanism for its superior handling of the Ds base.

Conclusions:

• The structural difference at residue Thr664 explains the high fidelity of Deep Vent DNA polymerase in replicating the Ds base.
• Understanding these polymerase-UBP interactions is key for designing novel DNA polymerases for synthetic biology.
• This study provides insights into enzyme engineering for expanding the biological information storage capacity.