Misincorporation and mispaired primer extension by human immunodeficiency virus reverse transcriptase

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Human immunodeficiency virus (HIV) reverse transcriptase (RT) is crucial for viral replication.
• Understanding RT fidelity is essential for developing antiviral therapies.
• Pre-steady-state kinetics provide insights into enzyme mechanisms.

Purpose of the Study:

• To investigate the fidelity mechanisms of HIV reverse transcriptase during DNA synthesis.
• To elucidate the kinetic parameters governing nucleotide incorporation and primer extension.
• To determine the impact of primer-template mismatches on RT activity.

Main Methods:

• Utilized pre-steady-state kinetic methods to analyze HIV reverse transcriptase activity.
• Measured enzyme kinetics for both complementary and non-complementary deoxynucleoside triphosphates (dNTPs).
• Assessed the influence of primer terminus pairing on nucleotide selection and extension rates.

Main Results:

• HIV RT exhibits reduced affinity for non-complementary dNTPs (1-2 orders of magnitude).
• The rate of the conformational change limiting nucleotide addition is reduced (1-4 orders of magnitude) for misincorporation.
• Primer terminus mismatches significantly reduce next dNTP affinity and extension rate, but extension of mispaired termini is still frequent.
• Mismatches can affect catalysis even when located 5' to the chemical step.

Conclusions:

• HIV RT fidelity is achieved through a combination of substrate discrimination and post-incorporation selection mechanisms.
• Primer terminus integrity is critical for accurate DNA synthesis by HIV RT.
• The enzyme's mechanism involves conformational changes and chemical steps that are sensitive to primer-template geometry.