THREONINE-435 OF ESCHERICHIA-COLI DNAA PROTEIN CONFERS SEQUENCE-SPECIFIC DNA-BINDING ACTIVITY

The Escherichia coil DnaA protein, as a sequence-specific DNA binding protein, promotes the initiation of chromosomal replication by binding to four asymmetric 9-mer sequences termed DnaA boxes in oriC. Charact erization of N terminal, C-terminal, and internal in-frame deletion mu tants identified residues near the C terminus of DnaA protein required for DNA binding. Furthermore, genetic and biochemical characterizatio n of 11 missense mutations mapping within the C-terminal 89 residues i ndicated that they were defective in DNA binding. Detailed biochemical characterization of one mutant protein bearing a threonine to methion ine substitution at position 435 (T435M) revealed that it retained onl y nonspecific DNA binding activity, suggesting that threonine 435 impa rts specificity in binding. Finally, T435M was inactive on its own for in vitro replication of an oriC plasmid but was able to augment limit ing levels of wild type DnaA protein, consistent with the proposal tha t not all of the DnaA monomers in the initial complex are bound specif ically to oriC and that direct interaction occurs among monomers.