Oligomeric state of the N-terminal domain of DnaT for replication restart in Escherichia coli.

DNA replication stops when chemical or physical damage occurs to the DNA. Repairing genomic DNA and reloading the replication helicase are crucial steps for restarting DNA replication. The Escherichia coli primosome is a complex of proteins and DNA responsible for reloading the replication helicase DnaB. DnaT, a protein found in the primosome complex, contains two functional domains. The C-terminal domain (89-179) forms an oligomeric complex with single-stranded DNA. Although the N-terminal domain (1-88) forms an oligomer, the specific residues responsible for this oligomeric structure have not yet been identified. In this study, we proposed that the N-terminal domain of DnaT has a dimeric antitoxin structure based on its primary sequence. Based on the proposed model, we confirmed the site of oligomerization in the N-terminal domain of DnaT through site-directed mutagenesis. The molecular masses and thermodynamic stabilities of the site-directed mutants located at the dimer interface, namely Phe42, Tyr43, Leu50, Leu53, and Leu54, were found to be lower than those of the wild-type. Moreover, we observed a decrease in the molecular masses of the V10S and F35S mutants compared to the wild-type DnaT. NMR analysis of the V10S mutant revealed that the secondary structure of the N-terminal domain of DnaT was consistent with the proposed model. Additionally, we have demonstrated that the stability of the oligomer formed by the N-terminal domain of DnaT is crucial for its function. Based on these findings, we propose that the DnaT oligomer plays a role in replication restart in Escherichia coli.

A structural model of the PriB-DnaT complex in Escherichia coli replication restart.

In Escherichia coli, DNA replication is restarted following DNA repair by the PriA-dependent pathway, in which the binding and dissociation of proteins such as PriA, PriB, and DnaT on ssDNA lead to the formation of a protein-DNA complex for recruiting the DnaB-DnaC replication protein complex. However, the structure of the PriB-DnaT complex, which is an essential step in the PriA-dependent pathway, remains elusive. In this study, the importance of His26 in PriB for replication restart was reconfirmed using plasmid complementation. Furthermore, we used NMR to examine the DnaT interaction sites on PriB. We also evaluated the PriB-DnaT peptide complex model, which was prepared by in silico docking, using molecular dynamic simulation. From these data, we propose a structural model that provides insight into the PriB-DnaT interaction.

Insight into the interaction between PriB and DnaT on bacterial DNA replication restart: Significance of the residues on PriB dimer interface and highly acidic region on DnaT.

When the replisome collapses at a DNA damage site, a sequence-independent replication restart system is required. In Escherichia coli, PriA, PriB, and DnaT assemble in an orderly fashion at the stalled replication fork and achieve the reloading of the replisome. PriB-DnaT interaction is considered a significant step in the replication restart. In this study, we examined the contribution of the residues Ser20, His26 and Ser55, which are located on the PriB dimer interface. These residues are proximal to Glu39 and Arg44, which are important for PriB-DnaT interaction. Mutational analyses revealed that His26 and Ser20 of PriB are important for the interaction with DnaT, and that the Ser55 residue of PriB might have a role in negatively regulating the DnaT binding. These residues are involved in not only the interaction between PriB and DnaT but also the dissociation of single-stranded DNA (ssDNA) from the PriB-ssDNA complex due to DnaT binding. Moreover, NMR study indicates that the region Asp66-Glu76 on the linker between DnaT domains is involved in the interaction with wild-type PriB. These findings provide significant information about the molecular mechanism underlying replication restart in bacteria.

Structure and mechanism of the primosome protein DnaT-functional structures for homotrimerization, dissociation of ssDNA from the PriB·ssDNA complex, and formation of the DnaT·ssDNA complex.

UNLABELLED: In Escherichia coli, the primosome plays an essential role in replication restart after dissociation of replisomes at the damaged replication fork. As well as PriA and PriB, DnaT, an ssDNA-binding protein, is a key member of the primosome. In this study, limited proteolysis indicated that E. coli DnaT was composed of two domains, consistent with the results of recent studies using Klebsiella pneumonia DnaT. We also found that a specific 24-residue region (Phe42-Asp66) in the N-terminal domain (1-88) was crucial for DnaT trimerization. Moreover, we determined the structure of the DnaT C-terminal domain (89-179) by NMR spectroscopy. This domain included three α-helices and a long flexible C-terminal tail, similar to the C-terminal subdomain of the AAA+ ATPase family. The neighboring histidines, His136 and His137, at a position corresponding to the AAA+ sensor II motif, were suggested to form an ssDNA-binding site. Furthermore, we found that the acidic linker between the two domains had an activity for dissociating ssDNA from the PriB·ssDNA complexes in a manner supported by the conserved acidic residues Asp70 and Glu76. Thus, these findings provide a novel structural basis for understanding the mechanism of DnaT in exposure of ssDNA and reloading of the replicative helicase at the stalled replication fork. DATABASE: The coordinates used for the ensemble of NMR structures have been deposited in the Protein Data Bank under accession code 2ru8. The NMR data have been deposited in the BioMagResBank (www.bmrb.wisc.edu) under accession number 11549.

Involvement of histidine in complex formation of PriB and single-stranded DNA.

PriB is a basic 10-kDa protein that acts as a facilitator in PriA-dependent replication restart in Escherichia coli. PriB has an OB-fold dimer structure and exhibits single-stranded DNA (ssDNA)-binding activities similar to single-stranded binding protein (SSB). In this study, we examined PriB's interaction with ssDNA (oligo-dT35, -dT15, and -dT7) using heteronuclear NMR analysis. Interestingly, (1)H or (15)N chemical shift changes of the PriB main-chain showed two distinct modes using oligo-dT35. The chemical shift perturbation sites in the primary mode were consistent with the main contact site in PriB-ssDNA, which was previously determined by crystal structure analysis. The results also suggested that approximately 8nt in ssDNA was the main contact site to PriB. In the secondary mode, residues in the α-helix region (His57-Ser65) and in ß4-loop3-ß5 were mainly perturbed. On the other hand, we examined the state of ssDNA by FRET using 5'-Cy3- and 3'-Cy5-modified oligo-dT35. As the PriB concentration increased, two-step saturation curves were observed in the FRET assay, suggesting a compact structure of ssDNA. Moreover, we confirmed two-step PriB binding to oligo-dT35 using EMSA. The pH dependence of FRET suggested contribution of the His residues. Therefore, we prepared His mutants of PriB and found that His64 in the α-helix region contributed to the second interaction between PriB and ssDNA using FRET and EMSA. Thus, from a structural standpoint, we suggested the role of His64 on the compactness of the PriB-ssDNA complex and on the positive cooperativity of PriB.

Measurement of evoked potential in recognition of faces and buildings.

In the functional neuro-imaging, it is known that the activation to the second stimulus is suppressed when two stimuli are given successively with a short interval as sensory inputs. This kind of suppressive phenomenon has been observed in event-related potential (ERP) signals as well as functional MRI signals of primary auditory, somatosensory and visual cortices. However, we rarely find reports to ERP suppression in higher-order areas of the brain. In this study we used a paired stimulus paradigm. The paired stimulus paradigm consisted of successively presented two stimuli in one trial. We recorded ERP related to recognition of faces and buildings to investigate the suppressive phenomenon in higher-order areas of the brain. We used the paired stimulus paradigm which was comprised of face, building and gray-colored-plain (gray) pictures. The inter-stimulus interval of two stimuli was 200 ms. On the points of O2 and T6, we observed that the ERP for the latter stimulus (face picture) was suppressed severely when a face-gray stimulus pair was presented. On the other hand, when a gray-building stimulus pair was presented, the ERP for the latter stimulus (building picture) was not suppressed on the points of O2 and T6. The similar suppression was observed with a face-face stimulus pair.