Abstract: Analysis of the functional domains of the replication initiator protein of the ColE2 plasmid by using temperature-sensitive mutantsThe replication initiator protein (Rep) of the ColE2 plasmid specifically binds to the replication origin (Ori).Rep synthesizes an RNA primer (5'-ppApGpA-3') at the unique site in Ori.It has been known that many enzymes synthesizing polynucleotides have active centers consisting of several acidic amino acids to trap a divalent metal ion to which a nucleotide binds.To elucidate the functional domains of the ColE2 Rep, we have constructed and analyzed amino acid residue deletion mutants and single amino acid residue substitution mutants.The DNA binding domain is located in the C-terminal region of Rep, and it has been suggested that the primase domain is located in the N-terminal region of Rep.Recently we examined the replication activity of these single amino acid residue substitution mutants at different temperatures and found that the mutant Rep proteins with D63A and E152A are inactive at 37˚C but active at 25˚C.These results suggest that these acidic amino acid residues are not involved in trapping a divalent metal ion but probably involved in formation of the structure of Rep.