Torsional control of double-stranded DNA branch migration

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA branched junctions are key intermediates in genetic recombination, mimicking Holliday junctions.
• Partially mobile junctions possess limited flanking homology, influencing branch point mobility.
• Understanding DNA junction dynamics is crucial for comprehending DNA repair and replication.

Purpose of the Study:

• To investigate the impact of superhelical torque on the position of branch points in partially mobile DNA junctions.
• To develop a synthetic DNA system for studying DNA junction mechanics under controlled conditions.
• To analyze how intercalating agents affect DNA junction structure and branch point location.

Main Methods:

• Construction of a synthetic double-stranded circular DNA molecule containing a partially mobile DNA branched junction.
• Incorporation of four homologous nucleotide pairs flanking the branch point, creating five potential branch locations.
• Analysis of branch point position using endonuclease VII cleavage in different topoisomers (relaxed and supercoiled) and in the presence of ethidium.

Main Results:

• The synthetic molecule formed two topoisomers upon ligation: a relaxed form and a form with one positive supercoil.
• In the relaxed topoisomer, the major branch point site was located at the maximally extruded position.
• Addition of ethidium caused the major branch point site to migrate to the minimally extruded position.

Conclusions:

• Superhelical torque influences the position of branch points in partially mobile DNA junctions.
• The synthetic DNA system effectively demonstrates torque-induced branch migration.
• Ethidium bromide acts as an intercalator, altering DNA conformation and consequently the branch point location.