Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• The Pup-proteasome system (PPS) is vital for mycobacteria, particularly in resisting reactive nitrogen intermediates (RNI).
• While pafA encodes a Pup-ligase, the functions of the adjacent pafB and pafC genes were unclear, with only a minor role suggested in RNI resistance.

Purpose of the Study:

• To elucidate the function of pafB and pafC in mycobacteria.
• To investigate the role of PafBC in DNA damage response pathways.

Main Methods:

• Generation of a pafBC deletion mutant in Mycobacterium smegmatis (Msm ΔpafBC).
• Proteome analysis to identify changes in protein levels.
• Assessing sensitivity to DNA damaging agents.
• Investigating transcriptional regulation of DNA repair genes, including recA.
• In vitro characterization of PafB and PafC protein interactions.

Main Results:

• Msm ΔpafBC exhibited decreased levels of DNA repair proteins, including RecA, and increased sensitivity to DNA damaging agents.
• PafB and PafC form a stable heterodimer with DNA binding motifs.
• Transcription of the recA gene via the P1 promoter was reduced in Msm ΔpafBC.
• Induction of recA expression following DNA damage was significantly impaired in the mutant.

Conclusions:

• PafBC acts as a heterodimeric transcriptional regulator in mycobacteria.
• PafBC plays a significant role in regulating the DNA damage response, particularly influencing recA expression.
• These findings reveal a novel regulatory function for PafBC in maintaining genomic stability in mycobacteria.