Footprint analysis of replicating murine leukemia virus reverse transcriptase

Area of Science:

• Molecular Biology
• Enzymology
• Virology

Background:

• Murine leukemia virus reverse transcriptase (MLV RT) is crucial for retroviral replication.
• Understanding the precise binding and protection mechanisms of RTs is essential for drug development.
• Previous structural models for DNA polymerases suggested different binding patterns.

Purpose of the Study:

• To visualize the replication complexes formed by wild-type MLV RT and a variant lacking RNase H activity (delta RH MLV RT).
• To determine the extent of template and primer nucleotide protection by these enzymes.
• To compare the binding characteristics and protection patterns with existing structural predictions.

Main Methods:

• Enzymatic footprinting was employed to map the regions of template and primer nucleotides protected by the enzymes.
• Experiments were conducted with wild-type MLV RT and a delta RH MLV RT variant.
• Binding affinities were assessed under varying ionic strength conditions.

Main Results:

• Wild-type MLV RT protected template nucleotides from +6 to -27 and primer nucleotides from -1 to -26.
• Delta RH MLV RT showed stable template-primer binding only at reduced ionic strength, protecting up to position -15.
• Both enzymes predominantly protected the template-primer duplex region, irrespective of hydrolysis profiles.

Conclusions:

• The binding and protection patterns of MLV RT and its delta RH variant primarily involve the template-primer duplex.
• These findings contradict recent structural predictions based on DNA polymerase beta.
• The RNase H domain influences stable binding but not the primary duplex protection site.