DNA polymerase III gene of Bacillus subtilis

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The DNA polymerase III holoenzyme is crucial for DNA replication in bacteria.
• The alpha subunit, encoded by the dnaF (polC) gene in Bacillus subtilis, is a key component of this enzyme.
• Understanding gene structure and mutations is vital for comprehending enzyme function and drug resistance mechanisms.

Purpose of the Study:

• To sequence the Bacillus subtilis dnaF (polC) gene, encoding the alpha subunit of DNA polymerase III holoenzyme.
• To identify the genetic basis of resistance to the antimicrobial drug 6-(p-hydroxyphenylazo)-uracil conferred by the azp-12 mutation.
• To investigate the functional implications of the mutation within the enzyme's active site and its homology to other DNA repair enzymes.

Main Methods:

• DNA sequencing of the Bacillus subtilis dnaF (polC) gene.
• Analysis of the azp-12 mutation, including base pair and amino acid changes.
• Bioinformatic analysis to compare amino acid composition with known enzyme subunits (e.g., E. coli epsilon subunit).

Main Results:

• The Bacillus subtilis dnaF gene sequence was determined: 4005 base pairs encoding 1335 amino acids.
• The azp-12 mutation involves a single base change (TCA to GCA at nucleotide 3523), substituting serine with alanine at position 1175.
• This altered amino acid is located in the enzyme's active site and C-terminal region; the N-terminal domain shows 26% homology to E. coli's epsilon subunit.

Conclusions:

• The study successfully sequenced the B. subtilis dnaF gene and pinpointed the mutation responsible for resistance to 6-(p-hydroxyphenylazo)-uracil.
• The findings support the role of the C-terminal active site in drug resistance and enzyme function.
• Homology to the E. coli epsilon subunit reinforces the integral nature of proofreading exonuclease activity within the B. subtilis DNA polymerase molecule.