Nucleotide-induced DNA polymerase active site motions accommodating a mutagenic DNA intermediate

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• DNA polymerases are crucial for DNA replication and repair.
• Errors in nucleotide incorporation by DNA polymerases can lead to mutations.
• The extension of DNA mismatches is a critical step in mutation formation.

Purpose of the Study:

• To elucidate the structural mechanisms by which DNA polymerase beta (pol beta) processes DNA mismatches.
• To understand how mismatches at the polymerase active site boundary influence nucleotide insertion.
• To provide insights into the fidelity of DNA replication and repair.

Main Methods:

• X-ray crystallography was used to determine the structures of DNA polymerase beta in complex with DNA substrates.
• Crystallographic structures were obtained for both binary complexes with mismatches and ternary complexes with incoming nucleotides.
• Structural analysis focused on the positioning of DNA elements and incoming nucleotides within the active site.

Main Results:

• A DNA mismatch at the polymerase active site boundary was characterized, showing specific base stacking and flipping interactions.
• Upon addition of dGTP, a ternary complex structure revealed the templating cytosine correctly positioned for Watson-Crick base pairing.
• Structural rearrangements, including primer terminus adenine rotation, were observed, but the 3'-hydroxyl group was mispositioned for efficient nucleotide insertion.

Conclusions:

• The structure of pol beta bound to a mismatch provides a molecular basis for understanding error extension.
• Specific conformational changes within the active site can accommodate mismatches but ultimately impede efficient extension.
• These findings contribute to understanding DNA repair mechanisms and the prevention of mutations.