Exonucleolytic proofreading by calf thymus DNA polymerase delta

Area of Science:

• Molecular Biology
• Enzymology
• Genetics

Background:

• DNA polymerase delta (pol delta) is crucial for DNA replication and repair.
• Understanding the fidelity of DNA polymerases is essential for assessing genome stability.
• Previous studies have indicated varying accuracy levels among different DNA polymerases.

Purpose of the Study:

• To determine the in vitro fidelity of calf thymus DNA polymerase delta (pol delta).
• To compare the accuracy of pol delta with DNA polymerases alpha and beta.
• To investigate the role of the associated 3' to 5' exonuclease in pol delta's fidelity.

Main Methods:

• Utilized an M13lacZ alpha nonsense codon reversion assay to measure DNA synthesis fidelity.
• Assessed the effect of deoxynucleoside triphosphate concentration on polymerase activity and exonuclease excision.
• Investigated the impact of adenosine monophosphate (AMP) on pol delta's polymerase and exonuclease activities.

Main Results:

• Pol delta demonstrated high fidelity, with an error rate below 1 single-base substitution per 10^6 nucleotides.
• Pol delta's accuracy was significantly higher (10- to 500-fold) than DNA polymerases alpha and beta.
• Evidence suggests proofreading by the 3' to 5' exonuclease contributes to pol delta's fidelity, as shown by reduced fidelity and excision with increased dNTPs and AMP inhibition.

Conclusions:

• Calf thymus DNA polymerase delta possesses a high degree of accuracy in DNA synthesis in vitro.
• The 3' to 5' exonuclease activity of pol delta plays a critical role in proofreading and enhancing DNA synthesis fidelity.
• These findings highlight the importance of proofreading mechanisms in maintaining genomic integrity by mammalian DNA polymerases.