Proofreading of ribonucleotides inserted into DNA by yeast DNA polymerase ɛ
Jessica S Williams1, Anders R Clausen, Stephanie A Nick McElhinny
1Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA.
Abstract:
We have investigated the ability of the 3' exonuclease activity of Saccharomyces cerevisiae DNA polymerase ɛ (Pol ɛ) to proofread newly inserted ribonucleotides (rNMPs). During DNA synthesis in vitro, Pol ɛ proofreads ribonucleotides with apparent efficiencies that vary from none at some locations to more than 90% at others, with rA and rU being more efficiently proofread than rC and rG. Previous studies show that failure to repair ribonucleotides in the genome of rnh201Δ strains that lack RNase H2 activity elevates the rate of short deletions in tandem repeat sequences. Here we show that this rate is increased by 2-4-fold in pol2-4 rnh201Δ strains that are also defective in Pol ɛ proofreading. In comparison, defective proofreading in these same strains increases the rate of base substitutions by more than 100-fold. Collectively, the results indicate that although proofreading of an 'incorrect' sugar is less efficient than is proofreading of an incorrect base, Pol ɛ does proofread newly inserted rNMPs to enhance genome stability.
Insights
Saccharomyces cerevisiae DNA polymerase ɛ proofreads newly inserted ribonucleotides, enhancing genome stability. Defective proofreading significantly increases mutation rates, particularly base substitutions.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Ribonucleotides (rNMPs) can be incorporated into DNA during synthesis.
- Accumulation of rNMPs can lead to genomic instability.
- RNase H2 is crucial for removing rNMPs, and its absence leads to increased short deletions.
Purpose of the Study:
- To investigate the role of Saccharomyces cerevisiae DNA polymerase ɛ (Pol ɛ) 3' exonuclease activity in proofreading rNMPs.
- To determine the impact of Pol ɛ proofreading defects on mutation rates in the absence of RNase H2.
Main Methods:
- In vitro DNA synthesis assays to measure Pol ɛ proofreading efficiency of different rNMPs.
- Analysis of mutation rates (short deletions and base substitutions) in yeast strains with defects in Pol ɛ proofreading and RNase H2.
Main Results:
- Pol ɛ exhibits variable efficiency in proofreading rNMPs, with higher efficiency for rA and rU compared to rC and rG.
- Deletion of RNase H2 (rnh201Δ) combined with a Pol ɛ proofreading defect (pol2-4) increased short deletion rates by 2-4 fold.
- The same combined defects led to a >100-fold increase in base substitution rates.
Conclusions:
- Pol ɛ's 3' exonuclease activity proofreads newly incorporated rNMPs, contributing to genome stability.
- Proofreading of incorrect sugar moieties by Pol ɛ is less efficient than proofreading incorrect bases.
- Defects in Pol ɛ proofreading significantly elevate mutation rates, highlighting its critical role in maintaining DNA integrity.
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