Bacillus subtilis polynucleotide phosphorylase 3'-to-5' DNase activity is involved in DNA repair

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Bacillus subtilis RecN protein exhibits ssDNA degradation activity in the presence of Mn(2+).
• This exonuclease activity was found not to be intrinsic to RecN itself.
• The study investigates the role of polynucleotide phosphorylase (PNPase) in this observed ssDNA degradation.

Purpose of the Study:

• To identify the enzyme responsible for ssDNA degradation in Bacillus subtilis.
• To elucidate the regulatory mechanisms and pathways involving PNPase in nucleic acid metabolism and DNA repair.

Main Methods:

• Purification of Bacillus subtilis RecN and PNPase.
• Enzyme activity assays using single-stranded DNA (ssDNA) and RNA substrates.
• Genetic analysis using various mutant strains (pnpA, recA, recN, ku, addA5, recO, recQ, recJ, recU, recG) to study epistasis and functional interactions.

Main Results:

• PNPase, in the presence of Mn(2+) and low inorganic phosphate (P(i)), degrades ssDNA with 3' --> 5' polarity.
• This ssDNA degradation is regulated by ATP and inhibited by Mg(2+) or high P(i), distinct from its Mg(2+)-dependent RNase activity.
• A null pnpA mutation affects DNA repair pathways, demonstrating PNPase's role in both RecA-dependent and RecA-independent repair.

Conclusions:

• PNPase possesses a distinct ssDNA exonuclease activity crucial for DNA repair.
• The regulation of PNPase's ssDNA and RNA degradation activities suggests mutually exclusive mechanisms.
• PNPase is implicated in diverse nucleic acid metabolic pathways and plays a significant role in bacterial DNA repair.