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

Abstract

DnaA 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 subtilis DnaD is needed during replication initiation for assembly of the replicative helicase at oriC and during replication restart at stalled replication forks. DnaD associates with DnaA at oriC and 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 regulate B. subtilis DnaA also affect DNA binding, whereas much of the regulation of Escherichia coli DnaA affects nucleotide hydrolysis or exchange. We found that the rate of nucleotide exchange for B. subtilis DnaA was high and not affected by DnaD. The rapid exchange is similar to that of Staphylococcus aureus DnaA and in contrast to the low exchange rate of Escherichia coli DnaA. 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.

The DnaA inhibitor SirA acts in the same pathway as Soj (ParA) to facilitate oriC segregation during Bacillus subtilis sporulation

“…Residues in this region of DnaA have also been implicated as possible sites of interaction with YabA and DnaD (Cho et al ., ; Scholefield and Murray, )(Fig. ), two other DnaA regulators that can also inhibit DnaA oligomerization (Bonilla and Grossman, ; Scholefield and Murray, ). It is feasible that if Soj targets this surface of DnaA, then substitutions that change the Soj‐DnaA interaction might also affect DnaD and YabA binding, leading to overreplication.…”

YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA

“…We then utilized one of these DnaA variants to show that YabA specifically inhibits DnaA helix formation in vitro . Moreover, based on the striking similarity of our results here regarding YabA, our previously reported results regarding Soj (Scholefield et al ., ), and a recent report regarding DnaD (Bonilla and Grossman, ), we have gone on to show that DnaD also inhibits DnaA helix formation in vitro . Taken together, these results indicate that all of the known regulators of B. subtilis DnaA present during vegetative growth specifically target DnaA helix formation during the initiation reaction.…”

New evidence of an old problem: The coupling of genome replication to cell growth in bacteria

“…It was recently indicated that the means of regulating replication initiation through YabA protein and pri mosome protein DnaS are similar by the mechanism. They both decrease the cooperativity of binding of DnaA with OriC and do not influence the ability of DnaA to bind or hydrolyze ATP [71,78].…”

“…Each of these mechanisms, according to our studies [43,44], is able to coordinate growth and replication in the cell by exponential type. These are the mechanisms regulat ing the DNA replication initiation process by YabA [70], DnaD [71], Soj [72], Spo0A, and SirA [5,[73][74][75][76] proteins. It is noteworthy that soj and yabA genes in B. subtilis are located near OriC, which is also in accordance with the mechanism of coordinating growth and replication suggested in the model [20].…”