Linearization and transposition of circular molecules of insertion sequence IS3

Area of Science:

• Molecular Biology
• Genetics
• DNA Transposition

Background:

• IS3 transposase facilitates the production of circular and linear IS3 DNA molecules from plasmids.
• IS3 circles contain the full IS3 sequence with terminal inverted repeats.
• Linear IS3 molecules exhibit specific nucleotide overhangs.

Purpose of the Study:

• To investigate the mechanism of IS3 circle and linear molecule formation.
• To determine the role of IS3 transposase in generating these DNA forms.
• To elucidate the pathway of IS3 transposition.

Main Methods:

• Plasmid construction with IS3 derivatives.
• Cloning and sequencing of generated DNA molecules.
• Site-directed mutagenesis of IS3 terminal sequences.
• Transposase cleavage assays.

Main Results:

• IS3 transposase generates IS3 circles and linear molecules even when flanking sequences differ.
• Linear molecules possess identical 5' protruding sequences, indicating a specific cleavage mechanism.
• Mutations in terminal dinucleotides reduce but do not abolish cleavage and transposition.
• IS3 circles are cleaved by transposase to form linear molecules, confirming a two-step process.
• Transposition of IS3 circles to target plasmids is dependent on terminal dinucleotides.

Conclusions:

• IS3 transposase generates linear IS3 molecules from IS3 circles via double-strand breaks at the intervening sequence.
• IS3 transposase exhibits dual cleavage activity, at the 3' end and near the 5' end.
• Terminal dinucleotides are crucial for both IS3 end cleavage and transposition.