Analysis of the DNA-binding domain of Escherichia coli DnaA protein

The DNA-binding domain of the Escherichia coli DnaA protein is represented by the 94 C-terminal amino acids (domain 4, aa 374-467). The isolated DNA-b inding domain acts as a functional repressor in vivo, as monitored with a m ioC::lacZ translational fusion integrated into the chromosome of the indica tor strain. In order to identify residues required for specific DNA binding , site-directed and random PCR mutagenesis were performed, using the mioC:: lacZ construct for selection. Mutations defective in DNA binding were found all over the DNA-binding domain with some clustering in the basic loop reg ion, within presumptive helix B and in a highly conserved region at the N-t erminus of presumptive helix C. Surface plasmon resonance (SPR) analysis re vealed different binding classes of mutant proteins. No or severely reduced binding activity was demonstrated for amino acid substitutions at position s R399, R407, Q408, H434, T435, T436 and A440. Altered binding specificity was found for mutations in a 12 residue region close to the N-terminus of h elix C. The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temp eratures. dnaX suppressors dnaA71 and dnaA721 map to the region close to he lix C and bind DNA non-specifically.