[Recombinant reverse transcriptase from Rous sarcoma virus. Kinetics and inhibition of DNA polymerase activity]

Area of Science:

• Molecular Biology
• Enzymology
• Virology

Background:

• Rous sarcoma virus reverse transcriptase is a key enzyme in retroviral replication.
• Understanding its enzymatic activity is vital for developing antiviral therapies.
• Previous studies have characterized its basic functions, but detailed kinetic mechanisms require further investigation.

Purpose of the Study:

• To elucidate the steady-state kinetics of DNA polymerization catalyzed by purified Rous sarcoma virus reverse transcriptase.
• To investigate the mechanism of inhibition of this enzyme's DNA-polymerase activity.
• To analyze the products of DNA synthesis to understand the role of the enzyme's subunits.

Main Methods:

• Purification of Rous sarcoma virus reverse transcriptase from E. coli HB101 (pMF14) to homogeneity.
• Enzymatic assays using poly(rA) as template, oligo(dT) as primer, and dTTP as substrate to determine kinetic constants.
• Studies on the inhibition of DNA-polymerase activity using various inhibitors.
• Analysis of the synthesized DNA products to characterize chain synthesis patterns.

Main Results:

• The enzyme exhibits steady-state kinetics consistent with an ordered reaction mechanism.
• Formation of a ternary complex occurs, with template-primer binding preceding substrate binding.
• Inhibitor studies provided insights into the enzyme's active site and mechanism.
• Analysis of reaction products indicated distribution synthesis of DNA chains by the alpha-subunit.

Conclusions:

• Rous sarcoma virus reverse transcriptase operates via an ordered kinetic mechanism involving ternary complex formation.
• The enzyme's DNA-polymerase activity can be modulated by specific inhibitors.
• The alpha-subunit plays a significant role in the distribution synthesis of DNA chains.