Excessive excision of correct nucleotides during DNA synthesis explained by replication hurdles

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA polymerases possess proofreading exonuclease activity to remove misincorporated nucleotides.
• This proofreading function is crucial for maintaining genomic integrity during DNA replication.

Purpose of the Study:

• To investigate the surprising high frequency of correctly incorporated nucleotide excision by the T7 replisome.
• To elucidate the mechanisms underlying excessive excision of correct nucleotides and its functional implications.

Main Methods:

• Utilized T7 replisome and two other DNA polymerases in in vitro replication assays.
• Analyzed nucleotide excision events during leading and lagging strand synthesis.
• Investigated the impact of replication hurdles like template secondary structures and helicase-polymerase uncoupling.

Main Results:

• T7 replisome excised approximately 7% of correctly incorporated nucleotides.
• This phenomenon was observed with other DNA polymerases, indicating generality.
• Replication hurdles induce DNA reannealing, polymerase backtracking, and frayed primer ends, leading to efficient exonuclease excision.
• Active-site shuttling occurs frequently and is not a byproduct of proofreading or degradation.

Conclusions:

• Replication stress significantly impacts DNA polymerase fidelity through excessive excision of correct nucleotides.
• Frequent active-site shuttling acts as a novel proofreading mechanism to safeguard primer ends.
• This mechanism prevents mutagenic extensions and ensures accurate DNA replication under challenging conditions.