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Study of MMLV RT- binding with DNA using surface plasmon resonance biosensor
Lei Wu1, Ming-Hui Huang, Jian-Long Zhao
1Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China.
Acta Biochimica Et Biophysica Sinica
|September 7, 2005
Summary
Moloney murine leukemia virus reverse transcriptase lacking the RNase H domain binds DNA, with affinity affected by inhibitors. Nevirapine enhanced binding, while efavirenz and quercetin showed minimal impact on Moloney murine leukemia virus reverse transcriptase-DNA interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Moloney murine leukemia virus reverse transcriptase (MMLV RT) is crucial for retroviral replication.
- The RNase H domain of MMLV RT plays a role in DNA binding and enzymatic activity.
- Understanding MMLV RT-DNA interactions is vital for developing antiviral therapies.
Purpose of the Study:
- To investigate the binding kinetics of MMLV RT lacking the RNase H domain (MMLV RT-) with various DNA substrates.
- To analyze the influence of specific inhibitors (efavirenz, nevirapine, quercetin) on MMLV RT--DNA binding.
- To elucidate the role of the RNase H domain in MMLV RT-DNA interactions.
Main Methods:
- Surface plasmon resonance (SPR) biosensor technique was employed for real-time binding analysis.
- Different DNA substrates (ssDNA, DNA template-primer duplex, gapped DNA) were immobilized on a biosensor chip.
- Kinetic parameters of MMLV RT--DNA binding were analyzed using Langmuir models in the presence and absence of inhibitors.
Main Results:
- MMLV RT- bound to single-stranded DNA (ssDNA) with associated conformational changes.
- Binding of MMLV RT- to DNA template-primer duplex and gapped DNA followed a 1:1 Langmuir model.
- The absence of the RNase H domain decreased the affinity between MMLV RT- and the DNA template-primer duplex.
- Nevirapine significantly increased MMLV RT--DNA binding affinity, primarily due to faster association and slower dissociation rates.
- Efavirenz showed a slight interference with MMLV RT--DNA binding, reducing affinity.
- Quercetin exhibited minimal effect on MMLV RT--DNA binding, despite its known inhibitory activity.
Conclusions:
- The RNase H domain is important for optimal affinity of MMLV RT to DNA template-primer duplexes.
- Nevirapine acts as a binding enhancer for MMLV RT- and DNA, suggesting a complex interaction mechanism.
- Efavirenz and quercetin have limited direct impact on the binding interaction between MMLV RT- and DNA, indicating their inhibitory mechanisms may target other aspects of RT function.