A native cruciform DNA structure probed in bacteria by recombinant T7 endonuclease

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• T7 endonuclease cleaves supercoiled plasmids at cruciform structures in vitro.
• In vivo, T7 endonuclease activity is competed by genomic single-stranded regions, limiting its effectiveness on plasmid vectors.
• Previous studies suggested cruciform structures might form in vivo but lacked direct evidence.

Purpose of the Study:

• To overcome competition for T7 endonuclease in vivo.
• To demonstrate the intracellular existence of cruciform DNA structures.
• To validate the use of endonucleases as probes for DNA topology in vivo.

Main Methods:

• Engineered a plasmid (pLAT75) with a more stable colE1 palindrome to form a robust cruciform structure.
• Increased plasmid copy number to enhance T7 endonuclease gene dosage and target size.
• Induction of T7 endonuclease and subsequent analysis of DNA degradation and plasmid modification.

Main Results:

• Induction of T7 endonuclease led to degradation of genomic DNA and intracellular nicking/linearization of the engineered plasmid.
• The cleavage site in vivo was mapped to the colE1 palindrome, specifically when it formed a cruciform structure.
• In vitro assays confirmed T7 and S1 endonuclease cleavage at the cruciform structure of the colE1 palindrome.

Conclusions:

• Cruciform DNA structures are present and accessible within the intracellular environment.
• Engineered plasmids and specific endonucleases can effectively probe and demonstrate in vivo DNA topology.
• This work validates the use of endonucleases as tools for studying DNA structures and dynamics within living cells.