Human DNA polymerase ε is able to efficiently extend from multiple consecutive ribonucleotides.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Replicative DNA polymerases (Pols) ensure replication fidelity by discriminating against mispaired nucleotides.
• Some yeast replicative Pols show low selectivity for deoxyribonucleotides over ribonucleotides.
• Ribonucleotides are abundant in human cells, suggesting potential genomic incorporation.

Purpose of the Study:

• To investigate the ability of human DNA polymerase ε (Pol ε) to incorporate ribonucleotides.
• To determine if human Pol ε can extend DNA synthesis from incorporated ribonucleotides.
• To assess the significance of ribonucleotide incorporation in human genomic DNA.

Main Methods:

• Biochemical assays using human DNA polymerase ε.
• Testing nucleotide incorporation and extension at physiological concentrations.
• Evaluating proofreading and exonuclease activity on ribonucleotide-containing primers.

Main Results:

• Human Pol ε readily incorporates ribonucleotides at physiological nucleotide concentrations.
• Nearly half of incorporated ribonucleotides escape proofreading by exonuclease-proficient Pol ε.
• Pol ε efficiently extends DNA synthesis from primers with up to five consecutive 3'-terminal ribonucleotides.
• Reduced exonuclease activity on ribonucleotide-containing primers contributes to efficient extension.

Conclusions:

• Human Pol ε's biochemical properties suggest it is a significant source of ribonucleotides in human genomic DNA.
• The low selectivity and efficient extension indicate that ribonucleotide incorporation is a likely event during normal cellular functions.
• These findings highlight a potential mechanism for genomic instability and disease.