THE A184V MISSENSE MUTATION OF THE DNAA5 AND DNAA46 ALLELES CONFERS ADEFECT IN ATP BINDING AND THERMOLABILITY IN INITIATION OF ESCHERICHIA-COLI DNA-REPLICATION

The temperature-sensitive dnaA5 and dnaA46 alleles each contain two mi ssense mutations. These mutations have been separated and the resultin g mutant proteins studied with regard to their role in initiation of D NA replication in vitro. Whereas the His-252 to tyrosine substitution (H252Y) unique to the dnaA46 allele did not affect the activities of D naA protein, the unique substitution of the dnaA5 allele, Gly-426 to s erine (G426S), was reduced in ifs DNA-binding affinity for oriC, the c hromosomal origin. This suggests that the C-terminal region of the Dna A protein is involved in DNA binding. The alanine-to-valine substituti on at amino acid 184 (A184V) that is common to both of the alleles is responsible for the thermolabile defect and lag in DNA synthesis of th ese mutants. Mutant proteins bearing the common substitution were defe ctive in ATP binding and were inactive in a replication system reconst ituted with purified proteins. DnaK and GrpE protein activated these m utant proteins for replication and ATP binding; the latter was measure d indirectly by the ATP-dependent formation of a trypsin-resistant pep tide. However, with this assay, the ATP-binding affinity appeared to b e reduced relative to wild-type DnaA protein. Activation was by conver sion of a self-aggregate to the monomer, and also by a conformational alteration that correlated with ATP binding.