Regulation in repressor inactivation by RecA protein

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA damage or inhibition of DNA synthesis in E. coli induces SOS functions.
• SOS functions are crucial for DNA repair, mutagenesis, cell division arrest, and prophage induction.
• Induction is triggered by LexA or phage repressor inactivation, mediated by E. coli RecA protein.

Purpose of the Study:

• To investigate the mechanisms controlling RecA protein-mediated repressor cleavage in vitro.
• To elucidate the roles of RecA protein structure and repressor interactions in SOS function induction.
• To identify the active site involved in repressor cleavage.

Main Methods:

• In vitro analysis of RecA protein-mediated cleavage of LexA and phage repressors.
• Investigation of RecA protein structural changes upon binding with single-stranded DNA (ssDNA) and ATP-gamma-S.
• Examination of the effect of C-terminal deletions on RecA protein activity.
• Analysis of repressor cleavage stimulation by specific DNA sequences (d(G-G)) and characterization of repressor fragments.

Main Results:

• RecA protein cleavage activity is regulated by its structural state within a ternary complex (RecA-ssDNA-ATP-gamma-S).
• RecA protein binding to ATP-gamma-S before ssDNA results in significantly reduced cleavage activity.
• Deletion of the C-terminal 25 amino acids constitutively activates RecA protein for repressor cleavage.
• Cleavage of phi 80cI repressor is stimulated by d(G-G), which binds to the repressor's C-terminal half.
• The C-terminal fragment of cleaved 80cI repressor exhibits self-cleavage activity.

Conclusions:

• RecA protein's C-terminal domain negatively controls its activation for repressor cleavage.
• The C-terminal domain of the repressor contains the active site for cleavage.
• The C-terminal fragment generated during cleavage can act as a nuclease, further processing the repressor.