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Intermolecular triplex formation distorts the DNA duplex in the regulatory region of human papillomavirus type-11
D A Hartman1, S R Kuo, T R Broker
1Department of Biochemistry, School of Medicine, University of Alabama, Birmingham 35294.
Abstract:
A conformational distortion in the DNA duplex at the regulatory region of human papillomavirus type-11 next to an intermolecular triplex, formed with a synthetic oligonucleotide, was investigated with several chemical probes. The sequence targeted for triplex formation borders on the binding sites for the regulatory proteins encoded by the viral E2 open reading frame. Dimethyl sulfate, diethyl pyrocarbonate, and OsO4 all react to a greater extent with nucleotides in the duplex that are immediately adjacent to the triplex as compared to other bases throughout the duplex. This hypermodification was observed on both the polypurine and polypyrimidine strands of the duplex DNA. Similar hyperreactivity of bases flanking a triplex also was seen when the contiguous target polypurine tract was effectively extended by mutating interrupting pyrimidines in the human papillomavirus type-11 sequence to purines. We propose that this hyperreactivity is due to a structural distortion caused by the junction between the triplex and the duplex tracts.
Insights
Chemical probes reveal DNA structural distortions at the junction of a human papillomavirus type-11 regulatory region and a synthetic oligonucleotide triplex. This DNA duplex hypermodification highlights conformational changes near viral regulatory protein binding sites.
Area of Science:
- Molecular Biology
- Structural Biology
- Virology
Background:
- Human papillomavirus type-11 (HPV-11) regulatory regions are crucial for viral gene expression.
- Intermolecular DNA triplexes can form in regulatory sequences, potentially altering DNA structure.
- Understanding DNA conformation near regulatory elements is key to deciphering viral replication and control.
Purpose of the Study:
- To investigate conformational distortions in the HPV-11 DNA duplex adjacent to an intermolecular triplex.
- To determine the impact of triplex formation on DNA accessibility and reactivity.
- To correlate DNA structural changes with viral regulatory protein binding sites.
Main Methods:
- Formation of an intermolecular triplex using a synthetic oligonucleotide and the HPV-11 regulatory region.
- Chemical probing using dimethyl sulfate, diethyl pyrocarbonate, and OsO4 to assess nucleotide accessibility.
- Site-directed mutagenesis to extend the polypurine tract and evaluate effects on triplex formation and reactivity.
Main Results:
- Chemical probes showed significantly increased reactivity of nucleotides in the DNA duplex bordering the triplex.
- This hypermodification occurred on both polypurine and polypyrimidine strands of the duplex DNA.
- Similar hyperreactivity was observed when the polypurine tract was extended via mutagenesis.
Conclusions:
- The junction between the DNA duplex and the intermolecular triplex induces a significant structural distortion.
- This distortion leads to enhanced chemical reactivity of adjacent nucleotides in the HPV-11 regulatory region.
- These findings suggest that triplex formation can alter DNA conformation in critical viral regulatory areas.