Multiple mechanisms for overcoming lethal over-initiation of DNA replication

DNA replication is a highly regulated process that is primarily controlled at the step of initiation. In the gram-positive bacterium Bacillus subtilis the replication initiator DnaA, is regulated by YabA, which inhibits cooperative binding at the origin. Mutants lacking YabA have increased and asynchronous initiation. We found that under conditions of rapid growth, the dnaA1 mutation that causes replication over-initiation, was synthetic lethal with a deletion of yabA. We isolated several classes of suppressors of the lethal phenotype of the ΔyabA dnaA1 double mutant. Some suppressors (dnaC, cshA) caused a decrease in replication initiation. Others (relA, nrdR) stimulate replication elongation. One class of suppressors decreased levels of the replicative helicase, DnaC, thereby limiting replication initiation. We found that decreased levels of helicase were sufficient to decrease replication initiation under fast growth conditions. Our results highlight the multiple mechanisms cells use to regulate DNA replication.