Chromosomal replication initiator protein dnaA | |||||||
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Identifiers | |||||||
Organism | |||||||
Symbol | DnaA | ||||||
Entrez | 948217 | ||||||
RefSeq (Prot) | NP_418157.1 | ||||||
UniProt | P03004 | ||||||
Other data | |||||||
Chromosome | genome: 3.88 - 3.88 Mb | ||||||
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Bac_DnaA_C | |||||||||
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Identifiers | |||||||||
Symbol | Bac_DnaA_C | ||||||||
Pfam | PF08299 | ||||||||
Pfam clan | CL0123 | ||||||||
InterPro | IPR013159 | ||||||||
SCOP2 | 1j1v / SCOPe / SUPFAM | ||||||||
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DnaA is a protein that activates initiation of DNA replication in bacteria. [1] Based on the Replicon Model, a positively active initiator molecule contacts with a particular spot on a circular chromosome called the replicator to start DNA replication. [2] It is a replication initiation factor which promotes the unwinding of DNA at oriC. [1] The DnaA proteins found in all bacteria engage with the DnaA boxes to start chromosomal replication. In addition to the DnaA protein, its concentration, binding to DnaA-boxes, and binding of ATP or ADP, we will cover the regulation of the DnaA gene, the unique characteristics of the DnaA gene expression, promoter strength, and translation efficiency. [2] The onset of the initiation phase of DNA replication is determined by the concentration of DnaA. [1] DnaA accumulates during growth and then triggers the initiation of replication. [1] Replication begins with active DnaA binding to 9-mer (9-bp) repeats upstream of oriC. [1] Binding of DnaA leads to strand separation at the 13-mer repeats. [1] This binding causes the DNA to loop in preparation for melting open by the helicase DnaB. [1]
Bac_DnaA | |||||||||
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Identifiers | |||||||||
Symbol | Bac_DnaA | ||||||||
Pfam | PF00308 | ||||||||
Pfam clan | CL0023 | ||||||||
InterPro | IPR013317 | ||||||||
PROSITE | PDOC00771 | ||||||||
SCOP2 | 1j1v / SCOPe / SUPFAM | ||||||||
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DnaA consists mainly in two different forms, the active ATP-form and the inactive ADP. [1] [3] The level of active DnaA within a cell is low immediately after a cell has divided. [1] Although the active form of DnaA requires ATP, the formation of the oriC/DnaA complex and subsequent DNA unwinding does not require ATP hydrolysis. [4]
The oriC site in E. coli has three A T rich 13 base pair regions (DUEs) followed by four 9 bp regions with the sequence TTAT(C or A)CA(C or A)A. [5] Around 10 DnaA molecules bind to the 9 bp regions, which wrap around the proteins causing the DNA at the AT-rich region to unwind. There are 8 DnaA binding sites within oriC, to which DnaA binds with differential affinity. When DNA replication is about to commence, DnaA occupies all of the high and low affinity binding sites. The denatured AT-rich region allows for the recruitment of DnaB (helicase), which complexes with DnaC (helicase loader). DnaC helps the helicase to bind to and to properly accommodate the ssDNA at the 13 bp region; this is accomplished by ATP hydrolysis, after which DnaC is released. Single-strand binding proteins (SSBs) stabilize the single DNA strands in order to maintain the replication bubble. DnaB is a 5'→3' helicase, so it travels on the lagging strand. It associates with DnaG (a primase) to form the only primer for the leading strand and to add RNA primers on the lagging strand. The interaction between DnaG and DnaB is necessary to control the longitude of Okazaki fragments on the lagging strand. DNA polymerase III is then able to start DNA replication.
DnaA is made up of four domains: the first is the N-terminal that associates with regulatory proteins, the second is a helical linker region, the third domain is a AAA+ region that binds to ATP, and the fourth domain is the C-terminal DNA binding region. [6] DnaA contains two conserved regions: the first is located in the central part of the protein and corresponds to the ATP-binding domain, the second is located in the C-terminal half and is involved in DNA-binding. [7]
The first strains to have the dnaA gene mutated were the temperature-sensitive K-12 strains CRT46 and CRT83, with the corresponding strain numbers beingdnaA46 and dnaA83. In contrary to dnaA mutants, the PC2 strain has a mutation in the dnaC gene, which codes for the loading factor for the DNA helicase dnaB. [8]
DnaA has the ability to bind its own promoter. When DnaA binds to its own promoter it blocks RNA polymerase from binding the promoter and inhibits initiation of transcription. In this way, DnaA is able to regulate its own expression. [3] [9] This process is called autoregulation. [10]
Each cell division cycle triggers a new round of chromosome replication by DnaA, the initiator protein. It is crucial to regulate DnaA-ATP monomer interactions with oriC during helicase loading and unwinding of origin DNA for precise timing. DnaA recognition sites in Escherichia coli are arranged in OriC to facilitate staged pre-replication complex assembling, with DnaA interacting with low affinity sites at it oligomerizes to fill the gaps between high affinity sites as it oligomerizes. There may be numerous gap-filling strategies to link OriC functions to bacterial lifestyles in nature, which may account for the wide variability of OriC DnaA recognition site patterns. [11] The two forms of DnaA, the active ATP- and ADP-form are regulated. The ATP-form is converted to the ADP-form through either Regulatory inactivation of DnaA (RIDA), [12] which in turn consists of the Hda protein and the β sliding clamp (DnaN) [13] and datA-dependent DnaA-ATP hydrolysis. [14] The ADP-form is converted to the ATP-form by DnaA-reactivating sequences 1 and 2 (DARS1 and DARS2). [15]
There are four disciplines within the DnaA protein. An initial comparison of Escherichia coli and Bacillus subtilis proteins led to the discovery of a sphere structure, which revealed a relatively conserved N-terminus and a largely conserved large C-terminus separated by a region that was mostly variable. [16] As an example, the Enterobacterial proteins have nearly identical N- and C-terminal sequences, however they are characterized by numerous amino acid adjustments, elisions, and insertions in the variable regions. [17] There is an AAA+ family ATPase motif and an independent DNA binding sphere in the C-terminal region. It was determined by NMR that Escherichia coli sphere IV had a crystal-clear structure when complexed with a DnaA- box. As a result, it was confirmed that the DNA list is intermediated by a combination of a helix-turn-helix motif and an introductory circle. When bound to ATP, but not to ADP, DnaA forms a super-helical structure with four monomers per turn. The structure of sphere I has been determined from three additional bacterial species and Escherichia coli by NMR. [18]
The research on dnaA(Ts) mutants provided the first proof that the dnaA gene is autoregulated. DnaA protein is still produced at non-permissive temperatures where it is inactive, but in some mutants it can be made active again by returning to a temperature that is conducive to development. [17] This reversible initiation capacity—which was larger than anticipated given the mass gain of the culture—could be seen in the absence of protein synthesis at the permissive temperature and suggested that the DnaA protein synthesis was derepressed at the high growth temperature. These results prompted a thorough investigation of the dnaA46 mutant under permissive, intermediate, and non-permissive development conditions. [19] The study's findings revealed that as growth temperature increased, the DnaA46 protein's activity decreased, leading to progressively decreasing DNA and origin concentrations at intermediate temperatures. An increase in initiation capacity was seen concurrently with a decrease in DnaA protein activity. Hansen and Rasmussen (1977) argued that the DnaA protein had a positive effect in replication initiation aing transcripts entering the dnaA gene were found as a result of sequencing the dnaA promoter region and the dnaA gene. [19] The DnaA promoter region has nine GATC sites within 225 base pairs, and a sequence that is similar to nd a negative role in its own synthesis based on these observations. Two promoters providrepetitions (DnaA-boxes) in the oriC region was found between the two promoters. According to several studies, the DnaA protein negatively regulates both promoters. In these research, it was discovered that the dnaA transcription was upregulated by 4- to 5-fold at non-permissive temperatures in dnaATs mutants and repressed by the same amount when DnaA protein was overproduced. The autoregulation of the dnaA gene requires the DnaA-box. [20] The sequence of the dnaA2p promoter region has some intriguing characteristics that can be seen more clearly. This promoter contains two GATC sites, one in the 10 sequence and the other in the 35 sequence, and both in vivo and in vitro, methylation increases transcription from this promoter by a factor of two. In addition, DnaA protein binds to regions upstream of the dnaA2p promoter with a high affinity. [10]
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part of biological inheritance. This is essential for cell division during growth and repair of damaged tissues, while it also ensures that each of the new cells receives its own copy of the DNA. The cell possesses the distinctive property of division, which makes replication of DNA essential.
Enterobacteria phage λ is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.
DnaB helicase is an enzyme in bacteria which opens the replication fork during DNA replication. Although the mechanism by which DnaB both couples ATP hydrolysis to translocation along DNA and denatures the duplex is unknown, a change in the quaternary structure of the protein involving dimerisation of the N-terminal domain has been observed and may occur during the enzymatic cycle. Initially when DnaB binds to dnaA, it is associated with dnaC, a negative regulator. After DnaC dissociates, DnaB binds dnaG.
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A pre-replication complex (pre-RC) is a protein complex that forms at the origin of replication during the initiation step of DNA replication. Formation of the pre-RC is required for DNA replication to occur. Complete and faithful replication of the genome ensures that each daughter cell will carry the same genetic information as the parent cell. Accordingly, formation of the pre-RC is a very important part of the cell cycle.
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A DNA unwinding element is the initiation site for the opening of the double helix structure of the DNA at the origin of replication for DNA synthesis. It is A-T rich and denatures easily due to its low helical stability, which allows the single-strand region to be recognized by origin recognition complex.
DNA adenine methylase, (Dam methylase) (also site-specific DNA-methyltransferase (adenine-specific), EC 2.1.1.72, modification methylase, restriction-modification system) is an enzyme that adds a methyl group to the adenine of the sequence 5'-GATC-3' in newly synthesized DNA. Immediately after DNA synthesis, the daughter strand remains unmethylated for a short time. It is an orphan methyltransferase that is not part of a restriction-modification system and regulates gene expression. This enzyme catalyses the following chemical reaction
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fis is an E. coli gene encoding the Fis protein. The regulation of this gene is more complex than most other genes in the E. coli genome, as Fis is an important protein which regulates expression of other genes. It is supposed that fis is regulated by H-NS, IHF and CRP. It also regulates its own expression (autoregulation). Fis is one of the most abundant DNA binding proteins in Escherichia coli under nutrient-rich growth conditions.
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T7 DNA polymerase is an enzyme used during the DNA replication of the T7 bacteriophage. During this process, the DNA polymerase “reads” existing DNA strands and creates two new strands that match the existing ones. The T7 DNA polymerase requires a host factor, E. coli thioredoxin, in order to carry out its function. This helps stabilize the binding of the necessary protein to the primer-template to improve processivity by more than 100-fold, which is a feature unique to this enzyme. It is a member of the Family A DNA polymerases, which include E. coli DNA polymerase I and Taq DNA polymerase.
Tus, also known as terminus utilization substance, is a protein that binds to terminator sequences and acts as a counter-helicase when it comes in contact with an advancing helicase. The bound Tus protein effectively halts DNA polymerase movement. Tus helps end DNA replication in prokaryotes.
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