The Cooperative Binding of the Replication Initiation Protein DnaA is Regulated by the Helicase Loader DnaD in Bacillus subtilis

Initiation of DNA replication requires binding of the initiator protein, DnaA, to the chromosomal origin of replication, oriC. In Bacillus subtilis and other low G+C content Gram-positive bacteria, the primosomal proteins DnaD and DnaB, in conjunction with loader DnaI, help load helicase at oriC.  DnaA also acts as a transcription factor at several sites outside of the oriC region, referred to as secondary sites. DnaD and DnaB are recruited to oriC and the secondary sites by their association with DnaA. The presence of DnaD and DnaB at the secondary targets of DnaA in the absence of helicase loading is suggestive of a role in modulating a general activity of DnaA. We tested the effect of DnaD on the cooperative nature of DnaA binding to multiple DNA targets in vitro. Using Electrophoretic Mobility Shift Assays (EMSA) and purified recombinant proteins, we observed that DnaD prevented the cooperative binding observed by DnaA alone, making it completely non-cooperative. Since cooperativity is important for replication, we propose that DnaD may inhibit DnaA’s ability to initiate replication and maybe even DnaA’s transcription factor functions. We are also testing the effect of the other replication protein DnaB as it is also found along DnaA and DnaD in vivo. Together, DnaD and DnaB may control DnaA’s activity by preventing re-initiation from oriC and/or transcription from DnaA controlled promoters.