THE SPEED OF THE ESCHERICHIA COIL FORK IN-VIVO DEPENDS ON THE DNAB-DNAC RATIO

The DnaC protein is required for loading the DnaB helicase at oriC. Th us DnaC promotes the formation of the pre-replication complex, but mus t leave the complex in order for the DnaB protein to function as a hel icase. In vitro, a slight excess of DnaC inhibits the movement of repl ication forks by inhibiting DnaB helicase activity (Allen and Kornberg , 1991). Here we show that inhibition of DNA replication by excess Dna C also occurs in vivo. The rate of replication-fork movement was measu red by flow cytometry. Initiation of replication was inhibited with ri fampicin and the rate of fork movement monitored during replication ru n-out by measuring the increase in the fraction of the cell population with fully replicated chromosomes. The replication rate was inversely related to the amount of excess DnaC protein. Initiation of replicati on was also inhibited. Co-overexpression of DnaB protein alleviated th e inhibition of replication caused by moderate excess of DnaC. The res ults show that DnaC interacts with replication forks during elongation in vivo, probably by binding to DnaB and inhibiting its helicase acti vity. Therefore, the ratio of DnaC to DnaB and the affinity of DnaC fo r a helicase hexamer at an established replication fork are of great i mportance for the rate of replication fork movement also in vivo.