Deoxynucleoside [1-thio]triphosphates prevent proofreading during in vitro DNA synthesis

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA polymerases are crucial for DNA replication and repair, with proofreading mechanisms enhancing their accuracy.
• Deoxynucleoside [1-thio]triphosphates (thio-dNTPs) are analogs used to study enzyme mechanisms.
• Proofreading involves a 3' to 5' exonucleolytic activity that removes misincorporated nucleotides.

Purpose of the Study:

• To quantify the contribution of proofreading to DNA polymerase fidelity using thio-dNTPs.
• To investigate the impact of thio-dNTP incorporation on DNA synthesis fidelity.
• To determine if proofreading can excise misincorporated thio-dNTPs.

Main Methods:

• Incorporation of thio-dNTPs into DNA using various DNA polymerases.
• Measurement of DNA synthesis fidelity using phi X174 am3 DNA sequencing.
• Comparison of fidelity between polymerases with and without proofreading activity.

Main Results:

• Thio-dNTPs were incorporated at rates similar to natural dNTPs.
• Fidelity was not affected by deoxycytidine [1-thio]triphosphate in polymerases lacking proofreading (avian myeloblastosis virus DNA polymerase, DNA polymerase beta).
• Fidelity was significantly reduced in polymerases with proofreading (E. coli DNA polymerase I, bacteriophage T4 DNA polymerase) when using deoxycytidine [1-thio]triphosphate, as these are not excised by the exonuclease.

Conclusions:

• Deoxynucleoside [1-thio]triphosphates possess normal base-pairing properties.
• Proofreading mechanisms are unable to excise misinserted thio-dNTPs.
• Proofreading contributes significantly to DNA polymerase fidelity, with E. coli DNA polymerase I fidelity increased 20-fold by proofreading a C:A mismatch.