REINITIATION KINETICS IN 8 DNAA(TS) MUTANTS OF ESCHERICHIA-COLI - RIFAMPICIN-RESISTANT INITIATION OF CHROMOSOME-REPLICATION

The kinetics of reinitiation of chromosome replication of eight dnaA(T s) mutants was investigated in an isogenic set of strains, Five mutant s (167, 46, 601, 606 and 5) are classified as reversible, since they c an reinitiate at 30 degrees C without protein synthesis, whereas the o ther three (508, 205, 204) require protein synthesis, In the presence of protein synthesis, reversible mutants initiate one round of replica tion rapidly after a shift to 30 degrees C, indicating that they conta in active or renaturable DnaA protein. The dnaA508 and dnaA204 mutants also reinitiate chromosome replication rapidly, whereas reinitiation is delayed 15-20 min in dnaA205. The dnaA508 and dnaA204 mutants might contain active DnaA protein just below the threshold level at 42 degr ees C and only require synthesis of small amounts of new DnaA protein before initiation at 30 degrees C, whereas dnaA205 accumulates DnaA pr otein for some time at 30 degrees C before reaching the initiation thr eshold. Three of the reversible mutants (5, 601, and 606) exhibited, i n addition to the protein synthesis-independent initiation capacity, a n RNA synthesis-independent initiation capacity. The thermal stability of these initiation capacities is the same as for mutant DnaA protein , strongly suggesting that mutant DnaA protein is responsible for both .