Replicative DNA polymerase δ but not ε proofreads errors in Cis and in Trans

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA polymerase ε (Pol ε) and DNA polymerase δ (Pol δ) are established as primary replicases for leading and lagging strands, respectively, in eukaryotes.
• The precise role of Pol δ in leading strand replication and its proofreading capabilities require further elucidation.

Purpose of the Study:

• To investigate the proofreading activity of Pol δ on the leading DNA strand.
• To determine the impact of proofreading-defective mutants of Pol ε and Pol δ on mutation rates in Saccharomyces cerevisiae.
• To explore the influence of gene orientation and specific DNA lesions on mutation rates.

Main Methods:

• Utilized a reporter system in diploid yeast strains (Saccharomyces cerevisiae) to quantify mutation rates at specific base pairs.
• Assessed the effects of heterozygous and homozygous proofreading-defective mutants of Pol ε and Pol δ.
• Analyzed mutation spectra, including AT→GC and CG→AT transitions, and the impact of 3'-terminal oxidative lesions.

Main Results:

• Wild-type Pol ε did not proofread errors generated by proofreading-defective Pol ε.
• Pol δ demonstrated proofreading activity against errors from both Pol δ and Pol ε.
• Gene orientation significantly affected mutation rates in proofreading-defective strains, attributed to mispair elongation efficiencies.
• A 3'-terminal 8-oxoG lesion opposite an adenine was efficiently extended and not proofread.

Conclusions:

• Pol δ plays a crucial role in proofreading leading strand synthesis, particularly when Pol ε function is impaired.
• The findings explain elevated mutation rates in Pol δ proofreading mutants and link DNA replication fidelity to cancer predisposition.
• Differential mispair elongation and resistance to proofreading of specific lesions contribute to mutation rate variations.