Restriction endonucleases can be used to study B-Z junctions in supercoiled DNA

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Z-DNA, a left-handed helical form of DNA, can form in sequences with alternating purine-pyrimidine repeats, such as (C-G)n.
• The formation of Z-DNA can alter DNA structure and affect interactions with proteins, including restriction endonucleases.
• Restriction enzymes recognize specific DNA sequences and cleave the DNA, but their activity can be modulated by local DNA conformation.

Purpose of the Study:

• To investigate the impact of Z-DNA formation on the cleavage activity of restriction endonucleases, specifically BamHI.
• To determine the influence of the B-Z DNA junction on enzyme inhibition.
• To define the spatial limits of structural perturbations associated with Z-DNA formation.

Main Methods:

• Construction of plasmids containing (C-G)n inserts within negatively supercoiled DNA.
• Enzymatic assays using restriction endonucleases BssHII and BamHI.
• Systematic variation of the position of the BamHI recognition site relative to the (C-G)n insert.
• Analysis of enzyme inhibition as a function of DNA superhelical density.

Main Results:

• The enzyme BssHII, recognizing G-C-G-C-G-C, showed strong inhibition when the (C-G)n insert adopted the Z-DNA conformation.
• Cleavage by BamHI was inhibited when its recognition sequence (G-G-A-T-C-C) was immediately adjacent to or four base pairs away from the Z-DNA forming insert.
• No significant inhibition of BamHI cleavage was observed when the recognition site was eight base pairs away from the Z-DNA insert.

Conclusions:

• Z-DNA formation in (C-G)n inserts can significantly inhibit the activity of restriction endonucleases like BamHI.
• The inhibitory effect of Z-DNA on BamHI cleavage is dependent on the proximity of the recognition site to the B-Z DNA junction.
• These findings help delineate the structural influence of Z-DNA and its associated B-Z junction on DNA-protein interactions.