A host-encoded DNA-binding protein promotes termination of plasmid replication at a sequence-specific replication...

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• DNA replication requires precise termination mechanisms to maintain genomic stability.
• The plasmid R6K possesses a replication terminus region known as tau.
• Understanding protein-DNA interactions at replication termini is key to deciphering replication control.

Purpose of the Study:

• To investigate the interaction between the Escherichia coli Ter protein and the R6K plasmid replication terminus (tau).
• To elucidate the role of the Ter protein-tau interaction in DNA replication termination.

Main Methods:

• Purification of the approximately 40-kDa Ter protein from Escherichia coli.
• Chemical footprinting assays to map Ter protein binding sites on the tau fragment.
• Site-directed mutagenesis of tau sequences to assess binding and functional consequences.
• In vivo analysis of DNA replication termination in the presence of mutated tau sequences.

Main Results:

• The Ter protein was purified with a 6600-fold enrichment.
• Ter protein specifically binds to two distinct sites within the 216-base-pair tau fragment.
• These binding sites exhibit inverted repeat sequences and are essential for Ter protein interaction.
• Mutagenesis of a specific tau sequence (tau R) abolished Ter protein binding and in vivo replication termination.

Conclusions:

• The Ter protein plays a critical role in the termination of DNA replication at the tau locus of the R6K plasmid.
• The specific binding of Ter protein to inverted repeat sequences within tau is essential for its function in replication termination.