Replication of ribonucleotide-containing DNA templates by yeast replicative polymerases

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Major DNA polymerases in Saccharomyces cerevisiae (Pols α, δ, and ɛ) incorporate ribonucleotides into DNA during synthesis.
• Unremoved ribonucleotides in the template strand can impede subsequent DNA replication efficiency and fidelity.

Purpose of the Study:

• To investigate the impact of ribonucleotides within DNA templates on the replication process.
• To determine the efficiency of DNA polymerases α, δ, and ɛ in bypassing single ribonucleotides.

Main Methods:

• Assessing the replication efficiency of Pols α, δ, and ɛ on DNA templates containing a single ribonucleotide.
• Evaluating the effect of dNTP concentrations and polymerase activity on bypass efficiency.

Main Results:

• All three polymerases (α, δ, and ɛ) demonstrated the ability to replicate past ribonucleotides.
• Bypass efficiency was slightly reduced compared to all-DNA templates, varying with ribo identity and sequence context.
• Bypass efficiencies for Pols δ and ɛ improved with increased dNTP levels and, for Pol ɛ, with inactivated 3'-exonuclease activity.

Conclusions:

• Ribonucleotides in DNA templates do not completely halt replication by Pols α, δ, and ɛ.
• The efficiency of bypassing ribonucleotides is comparable to or exceeds that of bypassing oxidative DNA lesions like 8-oxo-guanine.