ABSTRACT
Repair of DNA damage is vital to the health and survival of all organisms. In Escherichia coli, a protein known as RadA (or Sms) participates in recombinational repair, a process that uses an undamaged DNA strand in one DNA duplex to fill a gap in a homologous DNA strand in a sister DNA duplex. In a prior report, we described the production of monoclonal antibodies (MAbs) specific for RadA. Here, we investigated the epitopes recognized by two of the antibodies, MAbs 6F5 and 2A2. Premature stop codons (ochre mutations) were introduced into the radA gene at selected sites, and the truncated RadA proteins were probed by western blotting. Deletion of as few as four amino acids (457-460) from the C-terminus of RadA significantly increased the sensitivity of E. coli to ultraviolet (UV) radiation and abolished recognition of RadA by MAb 6F5. Single alanine substitutions made between positions 443-460 also adversely affected the ability of MAb 6F5 to bind to RadA, further supporting the idea that MAb 6F5 is specific for the RadA C-terminus. An ochre mutation at position 258 abolished the recognition of RadA by MAb 2A2, whereas an ochre mutation at position 279 did not, suggesting that MAb 2A2 binds to an epitope between residues 258 and 279. MAb 2A2 recognition of RadA was destroyed by endoproteinase glu-C cleavage of RadA at position 266, and by a single alanine substitution at position 265. In a competitive enzyme-linked immunosorbent assay (ELISA), a synthetic peptide comprising residues 263-273 of RadA blocked MAb 2A2 recognition of immobilized full-length RadA by more than 97%. We infer from our results that MAb 6F5 binds to the extreme C-terminus of RadA and that MAb 2A2 is specific for an epitope within positions 263-273.
