SITE-DIRECTED MUTATIONAL ANALYSIS FOR THE ATP BINDING OF DNAA PROTEIN- FUNCTIONS OF 2 CONSERVED AMINO-ACIDS (LYS-178 AND ASP-235) LOCATED IN THE ATP-BINDING DOMAIN OF DNAA PROTEIN IN-VITRO AND IN-VIVO

DnaA protein, the initiator of chromosomal DNA replication in Escheric hia coil, is activated by binding to ATP in vitro. We introduced site- directed mutations into two amino acids of the protein conserved among various ATP-binding proteins and examined functions of the mutated Dn aA proteins, in vitro and in vivo. Both mutated DnaA proteins (Lys-178 --> Ile or Asp-235 --> Asn) lost the affinity for both ATP and ADP bu t did maintain binding activity for oriC. Specific activities in an or iC DNA replication system in vitro were less than one-tenth those of t he wild-type protein. Assay of the generation of oriC sites sensitive to P1 nuclease, using the mutated DnaA proteins, revealed a defect in induction of the duplex opening at oriC. On the other hand, expression of each mutated DnaA protein in the temperature-sensitive dnaA46 muta nt did not complement the temperature sensitivity. We suggest that Lys -178 and Asp-235 of DnaA protein are essential for the activity needed to initiate oriC DNA replication in vitro and in vivo and that ATP bi nding to DnaA. protein is required for DNA replication-related functio ns.