Abstract
For bidirectional replication in Escherichia coli, higher order complexes are formed at the replication origin oriC by the initiator protein DnaA, which locally unwinds the left edge of oriC to promote the loading of two molecules of DnaB helicases onto the unwound region via dynamic interactions with the helicase-loader DnaC and the oriC-bound DnaA complex. One of the two helicases must translocate rightwards through oriC-bound DnaA complex. Here, we used a synthetic forked oriC DNA, which mimics the unwound state of oriC, to examine DnaB translocation through the oriC-bound DnaA complex. We found that DnaB helicase alone cannot pass through the oriC-bound DnaA complex without the help of single-strand binding protein (SSB). In the presence of SSB, DnaB passed through this complex along with its helicase function, releasing DnaA molecules. In addition, DnaB helicase activity is known to be inhibited by oversupply of DnaC, but this inhibition was relieved by SSB. These results suggest a mechanism that when two DnaB helicases are loaded at oriC, one translocates leftwards to expand the DnaA-unwound region and allows SSB binding to the single-stranded DNA, and such SSB molecules then stimulate translocation of the other helicase rightwards through the oriC-bound DnaA complex.