Autodigestion of lexA and phage lambda repressors

Area of Science:

• Molecular Biology
• Biochemistry
• Microbiology

Background:

• Proteolytic cleavage of the lexA repressor is crucial for activating the SOS DNA repair system in Escherichia coli.
• Previous studies suggested the recA protein directly catalyzes this lexA cleavage in vitro and in vivo.

Purpose of the Study:

• To investigate whether lexA repressor cleavage can occur independently of recA protein.
• To elucidate the mechanism and conditions influencing lexA repressor autodigestion.
• To compare lexA autodigestion with recA-dependent cleavage.

Main Methods:

• In vitro biochemical assays using highly-purified lexA protein.
• Analysis of cleavage products and reaction kinetics.
• Comparative studies with purified phage lambda repressor.

Main Results:

• Specific in vitro cleavage of lexA protein was observed in the absence of recA protein, indicating autodigestion.
• This autodigestion cleaved the same alanine-glycine bond as recA-dependent cleavage.
• Autodigestion was stimulated by alkaline pH and divalent metal ions (Ca2+, Co2+, Mg2+).
• Phage lambda repressor also exhibited similar, albeit slower, autodigestion.
• Autodigestion and recA-dependent cleavage share reaction pathways and site specificity.

Conclusions:

• The lexA repressor can catalyze its own specific cleavage.
• RecA protein likely acts as an indirect stimulatory factor (allosteric effector) rather than a direct protease in lexA cleavage.
• The findings suggest a shared reaction pathway for both autodigestion and recA-dependent cleavage of lexA.
• The protease and recA-recognition sites are located in the central or COOH-terminal region of the lexA protein.