DNA sequence requirements for replication fork arrest at terC in Bacillus subtilis.

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• The terC site in Bacillus subtilis is a specific chromosomal location where the clockwise DNA replication fork is halted.
• Understanding the DNA sequences and elements involved in replication termination is crucial for comprehending genome stability and replication dynamics.

Purpose of the Study:

• To investigate the role of DNA sequences flanking the terC site in Bacillus subtilis in mediating clockwise replication fork arrest.
• To elucidate the contribution of an adjacent open reading frame (ORF) and its regulatory elements to the terC-mediated fork arrest mechanism.

Main Methods:

• Deletional analysis of DNA sequences upstream and downstream of the terC site.
• Disruption of a specific open reading frame (ORF) adjacent to terC via insertion mutagenesis.
• Assessment of replication fork arrest at terC following genetic modifications.

Main Results:

• Deletion of DNA sequences up to 250 base pairs (bp) upstream of terC did not affect fork arrest, but further deletion of 130 bp abolished it.
• This critical upstream region contains an inverted repeat and potential promoter sequences for the adjacent ORF.
• Deletion of DNA within 80 bp downstream of terC, encompassing the majority of the ORF, also abolished fork arrest, as did disruption of the ORF itself.

Conclusions:

• Clockwise replication fork arrest at terC in Bacillus subtilis requires specific DNA sequences, including an upstream inverted repeat region.
• The protein product of the adjacent open reading frame (ORF) plays a critical role in the terC-mediated fork arrest mechanism.
• A model is proposed where both the DNA sequence features (inverted repeat) and the ORF protein product cooperate to arrest the replication fork at terC.