Characterization of the Chimeric PriB-SSBc Protein

PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein–protein interaction domain (SSBc). Apparently, the largest difference in structure between PriB and SSB is the lack of SSBc in PriB. In this study, we produced the chimeric PriB-SSBc protein in which Klebsiella pneumoniae PriB (KpPriB) was fused with SSBc of K. pneumoniae SSB (KpSSB) to characterize the possible SSBc effects on PriB function. The crystal structure of KpSSB was solved at a resolution of 2.3 Å (PDB entry 7F2N) and revealed a novel 114-GGRQ-117 motif in SSBc that pre-occupies and interacts with the ssDNA-binding sites (Asn14, Lys74, and Gln77) in SSBn. As compared with the ssDNA-binding properties of KpPriB, KpSSB, and PriB-SSBc, we observed that SSBc could significantly enhance the ssDNA-binding affinity of PriB, change the binding behavior, and further stimulate the PriA activity (an initiator protein in the pre-primosomal step of DNA replication), but not the oligomerization state, of PriB. Based on these experimental results, we discuss reasons why the properties of PriB can be retrofitted when fusing with SSBc.

Arresting chromosome replication upon energy starvation in Escherichia coli

Most organisms possess several cell cycle checkpoints to preserve genome stability in periods of stress. Upon starvation, the absence of chromosomal duplication in the bacterium Escherichia coli is ensured by holding off commencement of replication. During normal growth, accumulation of the initiator protein DnaA along with cell cycle changes in its activity, ensure that DNA replication starts only once per cell cycle. Upon nutrient starvation, the prevailing model is that an arrest in DnaA protein synthesis is responsible for the absence of initiation. Recent indications now suggest that DnaA degradation may also play a role. Here we comment on the implications of this potential new layer of regulation.

Putative Cooperative ATP–DnaA Binding to Double-Stranded DnaA Box and Single-Stranded DnaA-Trio Motif upon Helicobacter pylori Replication Initiation Complex Assembly

oriC is a region of the bacterial chromosome at which the initiator protein DnaA interacts with specific sequences, leading to DNA unwinding and the initiation of chromosome replication. The general architecture of oriCs is universal; however, the structure of oriC and the mode of orisome assembly differ in distantly related bacteria. In this work, we characterized oriC of Helicobacter pylori, which consists of two DnaA box clusters and a DNA unwinding element (DUE); the latter can be subdivided into a GC-rich region, a DnaA-trio and an AT-rich region. We show that the DnaA-trio submodule is crucial for DNA unwinding, possibly because it enables proper DnaA oligomerization on ssDNA. However, we also observed the reverse effect: DNA unwinding, enabling subsequent DnaA–ssDNA oligomer formation—stabilized DnaA binding to box ts1. This suggests the interplay between DnaA binding to ssDNA and dsDNA upon DNA unwinding. Further investigation of the ts1 DnaA box revealed that this box, together with the newly identified c-ATP DnaA box in oriC1, constitute a new class of ATP–DnaA boxes. Indeed, in vitro ATP–DnaA unwinds H. pylori oriC more efficiently than ADP–DnaA. Our results expand the understanding of H. pylori orisome formation, indicating another regulatory pathway of H. pylori orisome assembly.

Transcriptional Activity of the Bacterial Replication Initiator DnaA

In bacteria, DnaA is the most conserved DNA replication initiator protein. DnaA is a DNA binding protein that is part of the AAA+ ATPase family. In addition to initiating chromosome replication, DnaA can also function as a transcription factor either as an activator or repressor. The first gene identified to be regulated by DnaA at the transcriptional levels was dnaA. DnaA has been shown to regulate genes involved in a variety of cellular events including those that trigger sporulation, DNA repair, and cell cycle regulation. DnaA’s dual functions (replication initiator and transcription factor) is a potential mechanism for DnaA to temporally coordinate diverse cellular events with the onset of chromosome replication. This strategy of using chromosome replication initiator proteins as regulators of gene expression has also been observed in archaea and eukaryotes. In this mini review, we focus on our current understanding of DnaA’s transcriptional activity in various bacterial species.

Energy Starvation Induces a Cell Cycle Arrest in Escherichia coli by Triggering Degradation of the DnaA Initiator Protein

During steady-state Escherichia coli growth, the amount and activity of the initiator protein, DnaA, controls chromosome replication tightly so that initiation only takes place once per origin in each cell cycle, regardless of growth conditions. However, little is known about the mechanisms involved during transitions from one environmental condition to another or during starvation stress. ATP depletion is one of the consequences of long-term carbon starvation. Here we show that DnaA is degraded in ATP-depleted cells. A chromosome replication initiation block is apparent in such cells as no new rounds of DNA replication are initiated while replication events that have already started proceed to completion.

Sox14 is essential for initiation of interneuron differentiation in the chick spinal cord

The neural tube comprises several different types of progenitors and postmitotic neurons that coordinately act with each other to play integrated functions. Its development consists of two phases: proliferation of progenitor cells and differentiation into postmitotic neurons. How progenitor cells differentiate into each corresponding neuron is an important question for understanding the mechanisms of neuronal development. Here we introduce one of the Sox transcription factors, Sox14, which plays an essential role in the promotion of neuronal differentiation. Sox14 belongs to the SoxB subclass and its expression starts in the progenitor regions before neuronal differentiation is initiated at the trunk level of the neural tube. After neuronal differentiation is initiated, Sox14 expression gradually becomes confined to the V2a region of the neural tube, where Chx10 is co-expressed. Overexpression of Sox14 restricts progenitor cell proliferation. Conversely, the blockade of Sox14 expression by the RNAi strategy inhibits V2a neuron differentiation and causes expansion of the progenitor domain. We further found that Sox14 acted as a transcriptional activator. Taken together, Sox14 acts as a modulator of cell proliferation and an initiator protein for neuronal differentiation in the intermediate region of the neural tube.

AfsK-Mediated Site-Specific Phosphorylation Regulates DnaA Initiator Protein Activity in Streptomyces coelicolor

ABSTRACT In all organisms, chromosome replication is regulated mainly at the initiation step. Most of the knowledge about the mechanisms that regulate replication initiation in bacteria has come from studies on rod-shaped bacteria, such as Escherichia coli and Bacillus subtilis. Streptomyces is a bacterial genus that is characterized by distinctive features and a complex life cycle that shares some properties with the developmental cycle of filamentous fungi. The unusual lifestyle of streptomycetes suggests that these bacteria use various mechanisms to control key cellular processes. Here, we provide the first insights into the phosphorylation of the bacterial replication initiator protein, DnaA, from Streptomyces coelicolor. We suggest that phosphorylation of DnaA triggers a conformational change that increases its ATPase activity and decreases its affinity for the replication origin, thereby blocking the formation of a functional orisome. We suggest that the phosphorylation of DnaA is catalyzed by Ser/Thr kinase AfsK, which was shown to regulate the polar growth of S. coelicolor. Together, our results reveal that phosphorylation of the DnaA initiator protein functions as a negative regulatory mechanism to control the initiation of chromosome replication in a manner that presumably depends on the cellular localization of the protein. IMPORTANCE This work provides insights into the phosphorylation of the DnaA initiator protein in Streptomyces coelicolor and suggests a novel bacterial regulatory mechanism for initiation of chromosome replication. Although phosphorylation of DnaA has been reported earlier, its biological role was unknown. This work shows that upon phosphorylation, the cooperative binding of the replication origin by DnaA may be disturbed. We found that AfsK kinase is responsible for phosphorylation of DnaA. Upon upregulation of AfsK, chromosome replication occurred further from the hyphal tip. Orthologs of AfsK are exclusively found in mycelial actinomycetes that are related to Streptomyces and exhibit a complex life cycle. We propose that the AfsK-mediated regulatory pathway serves as a nonessential, energy-saving mechanism in S. coelicolor.