DNA tertiary structures formed in vitro by misaligned hybridization of multiple tandem repeat sequences

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA can adopt complex tertiary structures beyond the canonical double helix.
• Tandem repeat sequences are common in genomes and are associated with various genetic phenomena.
• Understanding DNA structural dynamics is crucial for comprehending gene regulation and disease.

Purpose of the Study:

• To investigate the formation of DNA tertiary structures in vitro from cloned DNA with tandem repeats.
• To characterize the structural properties and mapping of these novel DNA structures.
• To explore the potential of these structures as a method for identifying and mapping tandem repeat arrays.

Main Methods:

• Molecular cloning of DNA containing multiple tandem repeat sequences.
• Denaturation and reannealing of DNA in vitro.
• Electron microscopy for structural visualization and mapping.
• Analysis of DNA structures in homoduplex molecules and supercoiled plasmids.

Main Results:

• Knot-like DNA tertiary structures were observed in homoduplex DNA containing the human c-Harvey-ras gene's variable tandem repeat (VTR) sequence.
• These structures were mapped to the 812 bp VTR and are proposed to arise from slipped-strand mispairing and subsequent hybridization.
• Similar structures were found in linearized supercoiled plasmids, and more complex structures were observed in a 4 kb tandem array near the human alpha-globin locus.

Conclusions:

• DNA tertiary structures can be readily formed in vitro from cloned DNA containing tandem repeat sequences.
• The observed knot-like structures are a consequence of structural instabilities within tandem repeats.
• This in vitro formation method offers a practical approach for identifying and mapping direct tandem repeat arrays of at least 800 bp.