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1.
Escherichia coli DnaA is the initiator of chromosomal replication. Multiple ATP-DnaA molecules assemble at the oriC replication origin in a highly regulated manner, and the resultant initiation complexes promote local duplex unwinding within oriC, resulting in open complexes. DnaB helicase is loaded onto the unwound single-stranded region within oriC via interaction with the DnaA multimers. The tertiary structure of the functional domains of DnaA has been determined and several crucial residues in the initiation process, as well as their unique functions, have been identified. These include specific DNA binding, inter-DnaA interaction, specific and regulatory interactions with ATP and with the unwound single-stranded oriC DNA, and functional interaction with DnaB helicase. An overall structure of the initiation complex is also proposed. These are important for deepening our understanding of the molecular mechanisms that underlie DnaA assembly, oriC duplex unwinding, regulation of the initiation reaction, and DnaB helicase loading. In this review, we summarize recent progress on the molecular mechanisms of the functions of DnaA on oriC. In addition, some members of the AAA+ protein family related to the initiation of replication and its regulation (e.g., DnaA) are briefly discussed. 相似文献
2.
Xiaojing Li Yujiao Zhang Xintong Zhou Xinling Hu Yixuan Zhou Di Liu Anthony Maxwell Kaixia Mi 《Molecular microbiology》2019,111(6):1529-1543
3.
The DnaA inhibitor SirA acts in the same pathway as Soj (ParA) to facilitate oriC segregation during Bacillus subtilis sporulation
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DNA replication and chromosome segregation must be carefully regulated to ensure reproductive success. During Bacillus subtilis sporulation, chromosome copy number is reduced to two, and cells divide asymmetrically to produce the future spore (forespore) compartment. For successful sporulation, oriC must be captured in the forespore. New rounds of DNA replication are prevented in part by SirA, a protein that utilizes residues in its N‐terminus to directly target Domain I of the bacterial initiator, DnaA. Using a quantitative forespore chromosome organization assay, we show that SirA also acts in the same pathway as another DnaA regulator, Soj, to promote oriC capture in the forespore. By analyzing loss‐of‐function variants of both SirA and DnaA, we observe that SirA's ability to inhibit DNA replication can be genetically separated from its role in oriC capture. In addition, we identify substitutions near the C‐terminus of SirA and in DnaA Domain III that enhance interaction between the two proteins. One such variant, SirAP141T, remained functional in regard to inhibiting replication, but was unable to support oriC capture. Collectively, our results support a model in which SirA targets DnaA Domain I to inhibit DNA replication, and DnaA Domain III to facilitate Soj‐dependent oriC capture in the forespore. 相似文献
4.
The replication of chromosomal DNA is a fundamental event in the life cycle of every cell. The first step of replication, initiation, is controlled by multiple factors to ensure only one round of replication per cell cycle. The process of initiation has been described most thoroughly for bacteria, especially Escherichia coli, and involves many regulatory proteins that vary considerably between different species. These proteins control the activity of the two key players of initiation in bacteria: the initiator protein DnaA and the origin of chromosome replication (oriC). Factors involved in the control of the availability, activity, or oligomerization of DnaA during initiation are generally regarded as the most important and thus have been thoroughly characterized. Other aspects of the initiation process, such as origin accessibility and susceptibility to unwinding, have been less explored. However, recent findings indicate that these factors have a significant role. This review focuses on DNA topology, conformation, and methylation as important factors that regulate the initiation process in bacteria. We present a comprehensive summary of the factors involved in the modulation of DNA topology, both locally at oriC and more globally at the level of the entire chromosome. We show clearly that the conformation of oriC dynamically changes, and control of this conformation constitutes another, important factor in the regulation of bacterial replication initiation. Furthermore, the process of initiation appears to be associated with the dynamics of the entire chromosome and this association is an important but largely unexplored phenomenon. 相似文献
5.
In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes. 相似文献
6.
Rafał Donczew Thorsten Mielke Paweł Jaworski Jolanta Zakrzewska-Czerwińska Anna Zawilak-Pawlik 《Journal of molecular biology》2014
In bacteria, chromosome replication is initiated by binding of the DnaA initiator protein to DnaA boxes located in the origin of chromosomal replication (oriC). This leads to DNA helix opening within the DNA-unwinding element. Helicobacter pylori oriC, the first bipartite origin identified in Gram-negative bacteria, contains two subregions, oriC1 and oriC2, flanking the dnaA gene. The DNA-unwinding element region is localized in the oriC2 subregion downstream of dnaA. Surprisingly, oriC2–DnaA interactions were shown to depend on DNA topology, which is unusual in bacteria but is similar to initiator–origin interactions observed in higher organisms. In this work, we identified three DnaA boxes in the oriC2 subregion, two of which were bound only as supercoiled DNA. We found that all three DnaA boxes play important roles in orisome assembly and subsequent DNA unwinding, but different functions can be assigned to individual boxes. This suggests that the H. pylori oriC may be functionally divided, similar to what was described recently for Escherichia coli oriC. On the basis of these results, we propose a model of initiation complex formation in H. pylori. 相似文献
7.
Ariel Qui?ones Wolf-Rainer Jueterbock Walter Messer 《Molecular & general genetics : MGG》1991,231(1):81-87
Summary The initiation protein DnaA of Escherichia coli regulates its own expression autogenously by binding to a 9 by consensus sequence, the dnaA box, between the promoters dnaAP1 and dnaAP2. In this study, we analysed dnaA regulation in relation to DNA damage and found dnaA expression to be inducible by DNA lesions that inhibit DNA replication. On the other hand, coding DNA lesions were not able to induce dnaA expression. These results suggest that an additional regulatory mechanism is involved in dnaA gene expression and that DnaA protein may play a role in cellular responses to DNA damage. Furthermore, they strongly suggest that in response to DNA replication inhibition by DNA damage, and enhanced (re)initiation capacity is induced by oriC. 相似文献
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9.
The essential proteins DnaB, DnaD and DnaI of Bacillus subtilis are required for initiation, but not elongation, of DNA replication, and for replication restart at stalled forks. The interactions and functions of these proteins have largely been determined in vitro based on their roles in replication restart. During replication initiation in vivo, it is not known if these proteins, and the replication initiator DnaA, associate with oriC independently of each other by virtue of their DNA binding activities, as a (sub)complex like other loader proteins, or in a particular dependent order. We used temperature‐sensitive mutants or a conditional degradation system to inactivate each protein and test for association of the other proteins with oriC in vivo. We found that there was a clear order of stable association with oriC; DnaA, DnaD, DnaB, and finally DnaI‐mediated loading of helicase. The loading of helicase via stable intermediates resembles that of eukaryotes and the established hierarchy provides several potential regulatory points. The general approach described here can be used to analyse assembly of other complexes. 相似文献
10.
Rafa? Donczew Christoph Weigel Rudi Lurz Jolanta Zakrzewska-Czerwińska Anna Zawilak-Pawlik 《Nucleic acids research》2012,40(19):9647-9660
Binding of the DnaA protein to oriC leads to DNA melting within the DNA unwinding element (DUE) and initiates replication of the bacterial chromosome. Helicobacter pylori oriC was previously identified as a region localized upstream of dnaA and containing a cluster of DnaA boxes bound by DnaA protein with a high affinity. However, no unwinding within the oriC sequence has been detected. Comprehensive in silico analysis presented in this work allowed us to identify an additional region (oriC2), separated from the original one (oriC1) by the dnaA gene. DnaA specifically binds both regions, but DnaA-dependent DNA unwinding occurs only within oriC2. Surprisingly, oriC2 is bound exclusively as supercoiled DNA, which directly shows the importance of the DNA topology in DnaA-oriC interactions, similarly as previously presented only for initiator-origin interactions in Archaea and some Eukaryota. We conclude that H. pylori oriC exhibits bipartite structure, being the first such origin discovered in a Gram-negative bacterium. The H. pylori mode of initiator-oriC interactions, with the loop formation between the subcomplexes of the discontinuous origin, resembles those discovered in Bacillus subtilis chromosome and in many plasmids, which might suggest a similar way of controlling initiation of replication. 相似文献
11.
Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli 总被引:10,自引:0,他引:10
Tove Atlung Anders Løbner-Olesen Flemming G. Hansen 《Molecular & general genetics : MGG》1987,206(1):51-59
Summary Increased synthesis of DnaA protein, obtained with plasmids carrying the dnaA gene controlled by the heat inducible pL promoter, stimulated initiation of replication from oriC about threefold. The overinitiation was determined both as an increase in copy number of a minichromosome and as an increase in chromosomal gene dosage of oriC proximal DNA. The additional replication forks which were initiated on the chromosome did not lead to an overall increase in DNA content. DNA/DNA hybridization showed an amplification encompassing less than a few hundred kilobases on each side of oriC. Kinetic studies showed that the overinitiation occurred very rapidly after the induction, and that the initiation frequency then decreased to a near normal frequency per oriC. The results indicate that the DnaA protein is one important factor in regulation of initiation of DNA replication from oriC. 相似文献
12.
13.
14.
Abderrahim Malki Patrick Hughes Masamichi Kohiyama 《Molecular & general genetics : MGG》1991,225(3):420-426
Summary Heat shock proteins have been shown to be involved in many cellular processes in procaryotic and eucaryotic cells. Using an in vitro DNA replication assay, we show that DNA synthesis initiated at the chromosomal origin of replication of Escherichia coli (oriC) is considerably reduced in enzyme extracts isolated from cells bearing mutations in the dnaK and dnaJ genes, which code for heat shock proteins. Furthermore, unlike DNA synthesis in wild-type extracts, residual DNA synthesis in dnaK and dnaJ extracts is thermosensitive. Although thermosensitivity can be complemented by the addition of DnaK and DnaJ proteins, restoration of near wild-type replication levels requires supplementary quantities of purified DnaA protein. This key DNA synthesis initiator protein is shown to be adsorbed to DnaK affinity columns. These results suggest that at least one of the heat shock proteins, DnaK, exerts an effect on the initiation of DNA synthesis at the level of DnaA protein activity. However, our observation of normal oriC plasmid transformation ratios and concentrations in heat shock mutants at permissive temperatures would suggest that heat shock proteins play a role in DNA replication mainly at high temperatures or under other stressful growth conditions. 相似文献
15.
Mark J. Fogg Johan P. Turkenburg Anne Grahl Heath Murray Anthony J. Wilkinson 《Molecular microbiology》2014,93(5):975-991
Chromosome copy number in cells is controlled so that the frequency of initiation of DNA replication matches that of cell division. In bacteria, this is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA‐interacting protein that inhibits initiation of replication in diploid Bacillus subtilis cells committed to the developmental pathway leading to formation of a dormant spore. Here we present the crystal structure of SirA in complex with the N‐terminal domain of DnaA revealing a heterodimeric complex. The interacting surfaces of both proteins are α‐helical with predominantly apolar side‐chains packing in a hydrophobic interface. Site‐directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo. Localization of GFP–SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA. The SirA interacting surface of DnaA corresponds closely to the HobA‐interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor. 相似文献
16.
Gulpreet Kaur Mansi P. Vora Christopher A. Czerwonka Tania A. Rozgaja Julia E. Grimwade Alan C. Leonard 《Molecular microbiology》2014,91(6):1148-1163
During assembly of the E. coli pre‐replicative complex (pre‐RC), initiator DnaA oligomers are nucleated from three widely separated high‐affinity DnaA recognition sites in oriC. Oligomer assembly is then guided by low‐affinity DnaA recognition sites, but is also regulated by a switch‐like conformational change in oriC mediated by sequential binding of two DNA bending proteins, Fis and IHF, serving as inhibitor and activator respectively. Although their recognition sites are separated by up to 90 bp, Fis represses IHF binding and weak DnaA interactions until accumulating DnaA displaces Fis from oriC. It remains unclear whether high‐affinity DnaA binding plays any role in Fis repression at a distance and it is also not known whether all high‐affinity DnaA recognition sites play an equivalent role in oligomer formation. To examine these issues, we developed origin‐selective recombineering methods to mutate E. coli chromosomal oriC. We found that, although oligomers were assembled in the absence of any individual high‐affinity DnaA binding site, loss of DnaA binding at peripheral sites eliminated Fis repression, and made binding of both Fis and IHF essential. We propose a model in which interaction of DnaA molecules at high‐affinity sites regulates oriC DNA conformation. 相似文献
17.
DNA replication is a fundamental biological process that is tightly regulated in all cells. In bacteria, DnaA controls when and where replication begins by building a step‐wise complex that loads the replicative helicase onto chromosomal DNA. In many low‐GC Gram‐positive species, DnaA recruits the DnaD and DnaB proteins to function as adaptors to assist in helicase loading. How DnaA, its adaptors and the helicase form a complex at the origin is unclear. We addressed this question using the bacterial two‐hybrid assay to determine how the initiation proteins from Bacillus subtilis interact with each other. We show that cryptic interaction sites play a key role in this process and we map these regions for the entire pathway. In addition, we found that the SirA regulator that blocks initiation in sporulating cells binds to a surface on DnaA that overlaps with DnaD. The interaction between DnaA and DnaD was also mapped to the same DnaA surface in the human pathogen Staphylococcus aureus, demonstrating the broad conservation of this surface. Therefore, our study has unveiled key protein interactions essential for initiation and our approach is widely applicable for mapping interactions in other signaling pathways that are governed by cryptic binding surfaces. 相似文献
18.
Malgorzata Nowaczyk-Cieszewska Dorota Zyla-Uklejewicz Mateusz Noszka Pawel Jaworski Thorsten Mielke Anna Magdalena Zawilak-Pawlik 《Molecular microbiology》2020,113(2):338-355
The main roles of the DnaA protein are to bind the origin of chromosome replication (oriC), to unwind DNA and to provide a hub for the step-wise assembly of a replisome. DnaA is composed of four domains, with each playing a distinct functional role in the orisome assembly. Out of the four domains, the role of domain I is the least understood and appears to be the most species-specific. To better characterise Helicobacter pylori DnaA domain I, we have constructed a series of DnaA variants and studied their interactions with H. pylori bipartite oriC. We show that domain I is responsible for the stabilisation and organisation of DnaA-oriC complexes and provides cooperativity in DnaA–DNA interactions. Domain I mediates cross-interactions between oriC subcomplexes, which indicates that domain I is important for long-distance DnaA interactions and is essential for orisosme assembly on bipartite origins. HobA, which interacts with domain I, increases the DnaA binding to bipartite oriC; however, it does not stimulate but rather inhibits DNA unwinding. This suggests that HobA helps DnaA to bind oriC, but an unknown factor triggers DNA unwinding. Together, our results indicate that domain I self-interaction is important for the DnaA assembly on bipartite H. pylori oriC. 相似文献
19.
Summary Irradiation of Escherichia coli with UV light causes a transient inhibition of DNA replication. This effect is generally thought to be accounted for by blockage of the elongation of DNA replication by UV-induced lesions in the DNA (a cis effect). However, by introducing an unirradiated E. coli origin (oriC)-dependent replicon into UV-irradiated cells, we have been able to show that the environment of a UV-irradiated cell inhibits initiation of replication from oriC on a dimer-free replicon. We therefore conclude that UV-irradiation of E. coli leads to a trans-acting inhibition of initiation of replication. The inhibition is transient and does not appear to be an SOS function. 相似文献
20.
Control of DNA replication initiation is essential for cell growth. A unifying characteristic of DNA replication initiator proteins is their distinctive AAA+ nucleotide‐binding domains. The bacterial initiator DnaA assembles into a right‐handed helical oligomer built upon interactions between neighbouring AAA+ domains to form an active initiation complex. Recently we developed a unique cross‐linking assay that specifically detects ATP‐dependent DnaA helix assembly. Here we have utilized this assay to show that two DnaA regulatory proteins in Bacillus subtilis, YabA and DnaD, inhibit DnaA helix formation. These results, in combination with our previous finding that the regulatory factor Soj/ParA also targets DnaA filament formation, highlight the critical importance of regulating DnaA helix formation during the initiation reaction. Moreover, these observations lead us to suggest that DnaA oligomerization may be the main regulatory step of the initiator assembly pathway in B. subtilis, in contrast to the prevailing model of bacterial DNA replication based on Escherichia coli DnaA where ATP binding appears to be the targeted activity. 相似文献