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Retroviral integration into minichromosomes in vitro
P M Pryciak1, A Sil, H E Varmus
1Department of Biochemistry & Biophysics, University of California, San Francisco 94143-0502.
The EMBO Journal
|January 1, 1992
Summary
Retroviral integration targets chromatin, with murine leukemia virus (MLV) showing preferential integration into nucleosomes over naked DNA. This suggests integration machinery accesses exposed DNA on the nucleosome surface.
Area of Science:
- Molecular Biology
- Virology
- Epigenetics
Background:
- Retroviral integration is a critical step in viral replication.
- Understanding target site selection is key to controlling viral insertion and potential mutagenesis.
- Chromatin structure influences DNA accessibility and protein binding.
Purpose of the Study:
- To investigate chromatin as a target for retroviral integration in vitro.
- To determine if retroviral integration machinery exhibits preference for specific chromatin regions.
- To elucidate the mechanism of target site selection during retroviral DNA integration.
Main Methods:
- In vitro retroviral integration assays using murine leukemia virus (MLV).
- Employing yeast TRP1ARS1 and SV40 minichromosomes as model chromatin targets.
- Mapping integration sites via cloning and sequencing of recombinant DNA.
Main Results:
- Minichromosomes were integrated with efficiencies comparable to naked DNA.
- Under certain conditions, minichromosomes were preferentially targeted over naked DNA.
- Integration sites in chromatin showed ordered, periodic spacing (approx. 10 bp) and sequence bias.
- Integration machinery did not favor nucleosome-free or nuclease-sensitive regions.
Conclusions:
- Retroviral integration machinery likely accesses the exposed DNA helix face on nucleosomes.
- Chromatin structure limits and biases retroviral integration target sites.
- Integration site selection is influenced by DNA's rotational orientation within the nucleosome.