A thumb-domain insertion balances processivity and fidelity in DNA polymerase ε

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Cryo-EM structures reveal human DNA polymerase ε (Pol ε) interacting with PCNA via a Pol ε-specific thumb insertion (PTI).
• The PTI's proximity to PCNA suggests a regulatory role in DNA synthesis.

Purpose of the Study:

• To investigate the functional significance of the PTI and adjacent regions in yeast Pol ε.
• To elucidate the role of these domains in DNA replication fidelity and processivity.

Main Methods:

• Alanine-substitution variants were created in the yeast Pol ε thumb domain, targeting the PTI (polε-SLED) and adjacent loops (polε-PVTE, polε-KPFN).
• In vitro assays assessed polymerase processivity, DNA secondary structure bypass, and synthesis on long templates.
• In vivo mutator phenotypes were evaluated for the corresponding pol2 alleles.

Main Results:

• The polε-SLED variant showed increased intrinsic processivity and enhanced bypass of DNA secondary structures.
• A previous study with a larger substitution in the PTI region demonstrated reduced processivity, highlighting sensitivity to perturbation.
• polε-PVTE shifted activity towards exonuclease proofreading, while polε-KPFN showed increased sensitivity to secondary structures, partially rescued by PCNA.
• In vivo, pol2-SLED exhibited a modest mutator phenotype, while pol2-PVTE and pol2-KPFN showed minimal effects.

Conclusions:

• The PTI plays a crucial role in fine-tuning intrinsic processivity and proofreading during DNA replication.
• These regulatory functions are essential for maintaining replication fidelity during leading-strand synthesis.
• Subtle alterations in the PTI can lead to opposing functional outcomes, underscoring its delicate regulatory role.