Positive Charges Are Important for the SOS Constitutive Phenotype in recA730 and recA1202 Mutants of Escherichia coli K-12.

ABSTRACT

In Escherichia coli K-12, RecA binds to single-strand DNA (ssDNA) created by DNA damage to form a protein-DNA helical filament that serves to catalyze LexA autoproteolysis, which induces the SOS response. The SOS constitutive (SOSC) mutations recA730(E38K) and recA1202(Q184K) are both on the outside of the RecA filament, opposite to the face that binds DNA. recA730(E38K) is also able to suppress the UV sensitivity caused by recF mutations. Both SOSC expression and recF suppression are thought to be due to RecA730's ability to compete better for ssDNA coated with ssDNA-binding protein than the wild type. We tested whether other positively charged residues at these two positions would lead to SOSC expression and recF suppression. We found that 5/6 positively charged residues were SOSC and 4/5 of these were also recF suppressors. While other mutations at these two positions (and others) were recF suppressors, none were SOSC. Three recF suppressors could be made moderately SOSC by adding a recA operator mutation. We hypothesize two mechanisms for SOSC expression the first suggests that the positive charge at positions 38 and 184 attract negatively charged molecules that block interactions that would destabilize the RecA-DNA filament, and the second involves more stable filaments caused by increases in mutant RecA concentration. IMPORTANCE In Escherichia coli K-12, SOS constitutive (SOSC) mutants of recA turn on the SOS response in the absence of DNA damage. Some SOSC mutants are also able to indirectly suppress the UV sensitivity of recF mutations. Two SOSC mutations, recA730(E38K) and recA1202(Q184K), define a surface on the RecA-DNA filament opposite the surface that binds DNA. Both introduce positive charges, and recA730 is a recF suppressor. We tested whether the positive charge at these two positions was required for SOSC expression and recF suppression. We found a high correlation between the positive charge, SOSC expression and recF suppression. We also found several other mutations (different types) that provide recF suppression but no SOSC expression.