DNA polymerase β nucleotide-stabilized template misalignment fidelity depends on local sequence context.

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA polymerase β possesses two DNA-binding domains crucial for base excision repair.
• These domains interact with opposite sides of short DNA gaps, modifying DNA termini.
• Gaps larger than one nucleotide present challenges for these domains' interaction with DNA termini.

Purpose of the Study:

• To investigate the fidelity of DNA synthesis during the filling of 2-nucleotide DNA gaps.
• To determine the influence of local sequence context on DNA polymerase β activity in gapped DNA.
• To elucidate the structural and kinetic mechanisms underlying DNA synthesis fidelity in 2-nt gaps.

Main Methods:

• Crystallographic analysis of DNA polymerase β in complex with gapped DNA.
• Kinetic analyses of DNA synthesis across 2-nt gaps.
• Structural determination of a binary DNA product complex.

Main Results:

• DNA synthesis fidelity in 2-nt gaps is dependent on local DNA sequence context.
• Template dynamics alter which nucleotide in the gap serves as the coding nucleotide.
• Pyrimidine nucleotides in the initial coding position, particularly cytidine, decrease fidelity by allowing downstream template nucleotide coding.

Conclusions:

• DNA polymerase β can induce DNA strain, modulating coding nucleotide positioning and affecting incoming nucleotide selection.
• Template dynamics and sequence context introduce coding ambiguity, leading to errors even in "correct" DNA synthesis.
• Findings reveal a mechanism where DNA polymerase β's activity can lead to reduced fidelity in specific sequence contexts within 2-nt gaps.