Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
J Bacteriol. 2012 Sep;194(18):5110-7. doi: 10.1128/JB.00958-12. Epub 2012 Jul 20.
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.
DnaA 是一种 AAA+ATP 酶，也是细菌中保守的复制起始因子。细菌通过调节 DnaA 的活性来控制复制起始的时间。DnaA 结合到复制起点（oriC）中的多个位点，并且需要招募加载复制解旋酶所需的蛋白质。DnaA 还结合到其他染色体区域，并在这些区域中的一些作为转录因子发挥作用。枯草芽孢杆菌 DnaD 在复制起始时需要组装复制解旋酶在 oriC 处，并在停滞的复制叉处进行复制重启动。DnaD 在 oriC 处和 DnaA 结合的其他染色体区域与 DnaA 相关联。使用纯化的蛋白质，我们发现 DnaD 抑制了 DnaA 与 DNA 协同结合的能力，并导致表观解离常数降低。DnaD 的这些效应不依赖于 DnaA 结合或水解 ATP 的能力。已知其他调节枯草芽孢杆菌 DnaA 的蛋白质也影响 DNA 结合，而大肠杆菌 DnaA 的大部分调节作用则影响核苷酸水解或交换。我们发现枯草芽孢杆菌 DnaA 的核苷酸交换速率很高，不受 DnaD 的影响。这种快速交换类似于金黄色葡萄球菌 DnaA 的交换，与大肠杆菌 DnaA 的低交换率形成对比。我们认为，核苷酸交换速率高的 DnaA 主要调节 DnaA 的 DNA 结合活性，而核苷酸交换速率低的 DnaA 调节水解和交换。
