The Primosomal Protein DnaD Inhibits Cooperative DNA Binding by the Replication Initiator DnaA in Bacillus subtilis

Abstract

ABSTRACTDnaA is an AAA+ ATPase and the conserved replication initiator in bacteria. Bacteria control the timing of replication initiation by regulating the activity of DnaA. DnaA binds to multiple sites in the origin of replication (oriC) and is required for recruitment of proteins needed to load the replicative helicase. DnaA also binds to other chromosomal regions and functions as a transcription factor at some of these sites.Bacillus subtilisDnaD is needed during replication initiation for assembly of the replicative helicase atoriCand during replication restart at stalled replication forks. DnaD associates with DnaA atoriCand at other chromosomal regions bound by DnaA. Using purified proteins, we found that DnaD inhibited the ability of DnaA to bind cooperatively to DNA and caused a decrease in the apparent dissociation constant. These effects of DnaD were independent of the ability of DnaA to bind or hydrolyze ATP. Other proteins known to regulateB. subtilisDnaA also affect DNA binding, whereas much of the regulation ofEscherichia coliDnaA affects nucleotide hydrolysis or exchange. We found that the rate of nucleotide exchange forB. subtilisDnaA was high and not affected by DnaD. The rapid exchange is similar to that ofStaphylococcus aureusDnaA and in contrast to the low exchange rate ofEscherichia coliDnaA. We suggest that organisms in which DnaA has a high rate of nucleotide exchange predominantly regulate the DNA binding activity of DnaA and that those with low rates of exchange regulate hydrolysis and exchange.