High-affinity SV40 T-antigen binding sites in the human genome

Area of Science:

• Molecular Biology
• Genomics
• Virology

Background:

• The simian virus 40 (SV40) large T antigen is a viral protein with significant roles in viral replication and host cell transformation.
• Identifying specific DNA binding sites for viral proteins is crucial for understanding viral-host interactions and gene regulation.
• Human repetitive DNA elements, such as the L1 family, represent a substantial portion of the genome and their functions are not fully elucidated.

Purpose of the Study:

• To develop and apply methods for isolating human genomic DNA sequences with high-affinity binding sites for SV40 large T antigen.
• To characterize the identified DNA binding sites and their relationship to known SV40 binding sites or repetitive elements.
• To investigate the presence and behavior of SV40 T antigen binding sites within human repetitive DNA, particularly the L1 family.

Main Methods:

• Filter binding assay: SV40 T antigen was incubated with Sau3A-restricted human DNA, and T-antigen-DNA complexes were isolated using nitrocellulose filters.
• Chromatin immunoprecipitation: Nuclear chromatin from SV40-transformed human cells was fragmented, and T-antigen-bound fragments were immunoprecipitated.
• DNA cloning and sequencing: Isolated DNA fragments were cloned into plasmid vectors for further analysis and characterization of binding sites.

Main Results:

• The filter binding approach yielded four distinct human DNA fragments with high-affinity SV40 T antigen binding sites.
• One fragment contained a binding site similar to SV40's strong T-antigen binding site I; three others had multiple GA(G)GGC pentamers.
• Chromatin immunoprecipitation identified one fragment containing a T-antigen binding site, which was identified as belonging to the human L1 repetitive DNA family.

Conclusions:

• A significant portion of human L1 repetitive elements possess binding sites for SV40 T antigen.
• L1-related sequences were observed as extrachromosomal elements in an SV40-transformed human cell line.
• The quantity of extrachromosomal L1 DNA increased following the fusion of SV40-transformed cells with permissive monkey cells.