Function and biochemical characterization of RecJ in Deinococcus radiodurans

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• RecJ is a single-stranded DNA-specific nuclease crucial for double-stranded break (DSB) repair via the RecFOR pathway in bacteria.
• Deinococcus radiodurans is a bacterium known for its exceptional radiation resistance, with DSB repair primarily occurring through the RecFOR pathway.

Purpose of the Study:

• To investigate the specific role of the recJ gene in Deinococcus radiodurans.
• To characterize the biochemical properties and substrate specificity of the Deinococcus radiodurans RecJ (DrRecJ) protein.

Main Methods:

• Construction and analysis of a recJ null mutant in D. radiodurans.
• Expression and purification of the full-length DrRecJ protein.
• Biochemical assays to determine nuclease activity, substrate specificity, and regulatory factors.

Main Results:

• RecJ inactivation in D. radiodurans resulted in growth defects and sensitivity to high temperatures, but only a moderate decrease in radiation resistance.
• Purified DrRecJ exhibited Mn(2+)-dependent nuclease activity, with optimal activity at 0.1mM Mn(2+), suggesting Mn(2+) acts as a regulator.
• DrRecJ demonstrated broader substrate specificity for 5' ssDNA tails compared to E. coli RecJ, and its activity was modulated by D. radiodurans SSB (stimulation) and DdrB (inhibition).

Conclusions:

• The recJ gene is nonessential for the survival of D. radiodurans under tested conditions.
• DrRecJ's nuclease activity is regulated by manganese ions and interactions with SSB and DdrB proteins, highlighting a complex regulatory mechanism in D. radiodurans DNA repair.