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DNA replication

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.

DNA is made up of a double helix of two complementary strands. The double helix describes the appearance of a double-stranded DNA which is thus composed of two linear strands that run opposite to each other and twist together to form. During replication, these strands are separated. Each strand of the original DNA molecule then serves as a template for the production of its counterpart, a process referred to as semiconservative replication. As a result of semi-conservative replication, the new helix will be composed of an original DNA strand as well as a newly synthesized strand. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.

In a cell, DNA replication begins at specific locations, or origins of replication, in the genome which contains the genetic material of an organism. Unwinding of DNA at the origin and synthesis of new strands, accommodated by an enzyme known as helicase, results in replication forks growing bi-directionally from the origin. A number of proteins are associated with the replication fork to help in the initiation and continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new strands by adding nucleotides that complement each (template) strand. DNA replication occurs during the S-stage of interphase.

DNA replication (DNA amplification) can also be performed in vitro (artificially, outside a cell). DNA polymerases isolated from cells and artificial DNA primers can be used to start DNA synthesis at known sequences in a template DNA molecule. Polymerase chain reaction (PCR), ligase chain reaction (LCR), and transcription-mediated amplification (TMA) are examples. In March 2021, researchers reported evidence suggesting that a preliminary form of transfer RNA, a necessary component of translation, the biological synthesis of new proteins in accordance with the genetic code, could have been a replicator molecule itself in the very early development of life, or abiogenesis.

DNA structure
DNA exists as a double-stranded structure, with both strands coiled together to form the characteristic double helix. Each single strand of DNA is a chain of four types of nucleotides. Nucleotides in DNA contain a deoxyribose sugar, a phosphate, and a nucleobase. The four types of nucleotide correspond to the four nucleobases adenine, cytosine, guanine, and thymine, commonly abbreviated as A, C, G, and T. Adenine and guanine are purine bases, while cytosine and thymine are pyrimidines. These nucleotides form phosphodiester bonds, creating the phosphate-deoxyribose backbone of the DNA double helix with the nucleobases pointing inward (i.e., toward the opposing strand). Nucleobases are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine (two hydrogen bonds), and guanine pairs with cytosine (three hydrogen bonds).

DNA strands have a directionality, and the different ends of a single strand are called the "3′ (three-prime) end" and the "5′ (five-prime) end". By convention, if the base sequence of a single strand of DNA is given, the left end of the sequence is the 5′ end, while the right end of the sequence is the 3′ end. The strands of the double helix are anti-parallel, with one being 5′ to 3′, and the opposite strand 3′ to 5′. These terms refer to the carbon atom in deoxyribose to which the next phosphate in the chain attaches. Directionality has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3′ end of a DNA strand.

The pairing of complementary bases in DNA (through hydrogen bonding) means that the information contained within each strand is redundant. Phosphodiester (intra-strand) bonds are stronger than hydrogen (inter-strand) bonds. The actual job of the phosphodiester bonds is where in DNA polymers connect the 5' carbon atom of one nucleotide to the 3' carbon atom of another nucleotide, while the hydrogen bonds stabilize DNA double helices across the helix axis but not in the direction of the axis. This makes it possible to separate the strands from one another. The nucleotides on a single strand can therefore be used to reconstruct nucleotides on a newly synthesized partner strand.

DNA polymerase

DNA polymerases are a family of enzymes that carry out all forms of DNA replication. DNA polymerases in general cannot initiate synthesis of new strands but can only extend an existing DNA or RNA strand paired with a template strand. To begin synthesis, a short fragment of RNA, called a primer, must be created and paired with the template DNA strand.

DNA polymerase adds a new strand of DNA by extending the 3′ end of an existing nucleotide chain, adding new nucleotides matched to the template strand, one at a time, via the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from hydrolysis of the high-energy phosphate (phosphoanhydride) bonds between the three phosphates attached to each unincorporated base. Free bases with their attached phosphate groups are called nucleotides; in particular, bases with three attached phosphate groups are called nucleoside triphosphates. When a nucleotide is being added to a growing DNA strand, the formation of a phosphodiester bond between the proximal phosphate of the nucleotide to the growing chain is accompanied by hydrolysis of a high-energy phosphate bond with release of the two distal phosphate groups as a pyrophosphate. Enzymatic hydrolysis of the resulting pyrophosphate into inorganic phosphate consumes a second high-energy phosphate bond and renders the reaction effectively irreversible.

In general, DNA polymerases are highly accurate, with an intrinsic error rate of less than one mistake for every 107 nucleotides added. Some DNA polymerases can also delete nucleotides from the end of a developing strand in order to fix mismatched bases. This is known as proofreading. Finally, post-replication mismatch repair mechanisms monitor the DNA for errors, being capable of distinguishing mismatches in the newly synthesized DNA Strand from the original strand sequence. Together, these three discrimination steps enable replication fidelity of less than one mistake for every 109 nucleotides added.

The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected E. coli. During the period of exponential DNA increase at 37 °C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 108.

Replication process

DNA replication, like all biological polymerization processes, proceeds in three enzymatically catalyzed and coordinated steps: initiation, elongation and termination.

Initiation
For a cell to divide, it must first replicate its DNA. DNA replication is an all-or-none process; once replication begins, it proceeds to completion. Once replication is complete, it does not occur again in the same cell cycle. This is made possible by the division of initiation of the pre-replication complex.

Pre-replication complex
In late mitosis and early G1 phase, a large complex of initiator proteins assembles into the pre-replication complex at particular points in the DNA, known as "origins". In E. coli the primary initiator protein is Dna A; in yeast, this is the origin recognition complex. Sequences used by initiator proteins tend to be "AT-rich" (rich in adenine and thymine bases), because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair) and thus are easier to strand-separate. In eukaryotes, the origin recognition complex catalyzes the assembly of initiator proteins into the pre-replication complex. In addition, a recent report suggests that budding yeast ORC dimerizes in a cell cycle dependent manner to control licensing. In turn, the process of ORC dimerization is mediated by a cell cycle-dependent Noc3p dimerization cycle in vivo, and this role of Noc3p is separable from its role in ribosome biogenesis. An essential Noc3p dimerization cycle mediates ORC double-hexamer formation in replication licensing ORC and Noc3p are continuously bound to the chromatin throughout the cell cycle. Cdc6 and Cdt1 then associate with the bound origin recognition complex at the origin in order to form a larger complex necessary to load the Mcm complex onto the DNA. In eukaryotes, the Mcm complex is the helicase that will split the DNA helix at the replication forks and origins. The Mcm complex is recruited at late G1 phase and loaded by the ORC-Cdc6-Cdt1 complex onto the DNA via ATP-dependent protein remodeling. The loading of the Mcm complex onto the origin DNA marks the completion of pre-replication complex formation.

If environmental conditions are right in late G1 phase, the G1 and G1/S cyclin-Cdk complexes are activated, which stimulate expression of genes that encode components of the DNA synthetic machinery. G1/S-Cdk activation also promotes the expression and activation of S-Cdk complexes, which may play a role in activating replication origins depending on species and cell type. Control of these Cdks vary depending on cell type and stage of development. This regulation is best understood in budding yeast, where the S cyclins Clb5 and Clb6 are primarily responsible for DNA replication. Clb5,6-Cdk1 complexes directly trigger the activation of replication origins and are therefore required throughout S phase to directly activate each origin.

In a similar manner, Cdc7 is also required through S phase to activate replication origins. Cdc7 is not active throughout the cell cycle, and its activation is strictly timed to avoid premature initiation of DNA replication. In late G1, Cdc7 activity rises abruptly as a result of association with the regulatory subunit DBF4, which binds Cdc7 directly and promotes its protein kinase activity. Cdc7 has been found to be a rate-limiting regulator of origin activity. Together, the G1/S-Cdks and/or S-Cdks and Cdc7 collaborate to directly activate the replication origins, leading to initiation of DNA synthesis.

Preinitiation complex
In early S phase, S-Cdk and Cdc7 activation lead to the assembly of the preinitiation complex, a massive protein complex formed at the origin. Formation of the preinitiation complex displaces Cdc6 and Cdt1 from the origin replication complex, inactivating and disassembling the pre-replication complex. Loading the preinitiation complex onto the origin activates the Mcm helicase, causing unwinding of the DNA helix. The preinitiation complex also loads α-primase and other DNA polymerases onto the DNA.

After α-primase synthesizes the first primers, the primer-template junctions interact with the clamp loader, which loads the sliding clamp onto the DNA to begin DNA synthesis. The components of the preinitiation complex remain associated with replication forks as they move out from the origin.

Elongation
DNA polymerase has 5′–3′ activity. All known DNA replication systems require a free 3′ hydroxyl group before synthesis can be initiated (note: the DNA template is read in 3′ to 5′ direction whereas a new strand is synthesized in the 5′ to 3′ direction—this is often confused). Four distinct mechanisms for DNA synthesis are recognized:
 * 1) All cellular life forms and many DNA viruses, phages and plasmids use a primase to synthesize a short RNA primer with a free 3′ OH group which is subsequently elongated by a DNA polymerase.
 * 2) The retroelements (including retroviruses) employ a transfer RNA that primes DNA replication by providing a free 3′ OH that is used for elongation by the reverse transcriptase.
 * 3) In the adenoviruses and the φ29 family of bacteriophages, the 3′ OH group is provided by the side chain of an amino acid of the genome attached protein (the terminal protein) to which nucleotides are added by the DNA polymerase to form a new strand.
 * 4) In the single stranded DNA viruses—a group that includes the circoviruses, the geminiviruses, the parvoviruses and others—and also the many phages and plasmids that use the rolling circle replication (RCR) mechanism, the RCR endonuclease creates a nick in the genome strand (single stranded viruses) or one of the DNA strands (plasmids). The 5′ end of the nicked strand is transferred to a tyrosine residue on the nuclease and the free 3′ OH group is then used by the DNA polymerase to synthesize the new strand.

Cellular organisms use the first of these pathways since it is the most well-known. In this mechanism, once the two strands are separated, primase adds RNA primers to the template strands. The leading strand receives one RNA primer while the lagging strand receives several. The leading strand is continuously extended from the primer by a DNA polymerase with high processivity, while the lagging strand is extended discontinuously from each primer forming Okazaki fragments. RNase removes the primer RNA fragments, and a low processivity DNA polymerase distinct from the replicative polymerase enters to fill the gaps. When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. Ligase works to fill these nicks in, thus completing the newly replicated DNA molecule.

The primase used in this process differs significantly between bacteria and archaea/eukaryotes. Bacteria use a primase belonging to the DnaG protein superfamily which contains a catalytic domain of the TOPRIM fold type. The TOPRIM fold contains an α/β core with four conserved strands in a Rossmann-like topology. This structure is also found in the catalytic domains of topoisomerase Ia, topoisomerase II, the OLD-family nucleases and DNA repair proteins related to the RecR protein.

The primase used by archaea and eukaryotes, in contrast, contains a highly derived version of the RNA recognition motif (RRM). This primase is structurally similar to many viral RNA-dependent RNA polymerases, reverse transcriptases, cyclic nucleotide generating cyclases and DNA polymerases of the A/B/Y families that are involved in DNA replication and repair. In eukaryotic replication, the primase forms a complex with Pol α.

Multiple DNA polymerases take on different roles in the DNA replication process. In E. coli, DNA Pol III is the polymerase enzyme primarily responsible for DNA replication. It assembles into a replication complex at the replication fork that exhibits extremely high processivity, remaining intact for the entire replication cycle. In contrast, DNA Pol I is the enzyme responsible for replacing RNA primers with DNA. DNA Pol I has a 5′ to 3′ exonuclease activity in addition to its polymerase activity, and uses its exonuclease activity to degrade the RNA primers ahead of it as it extends the DNA strand behind it, in a process called nick translation. Pol I is much less processive than Pol III because its primary function in DNA replication is to create many short DNA regions rather than a few very long regions.

In eukaryotes, the low-processivity enzyme, Pol α, helps to initiate replication because it forms a complex with primase. In eukaryotes, leading strand synthesis is thought to be conducted by Pol ε; however, this view has recently been challenged, suggesting a role for Pol δ. Primer removal is completed Pol δ while repair of DNA during replication is completed by Pol ε.

As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming a replication fork with two prongs. In bacteria, which have a single origin of replication on their circular chromosome, this process creates a "theta structure" (resembling the Greek letter theta: θ). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these.

Replication fork
The replication fork is a structure that forms within the long helical DNA during DNA replication. It is produced by enzymes called helicases that break the hydrogen bonds that hold the DNA strands together in a helix. The resulting structure has two branching "prongs", each one made up of a single strand of DNA. These two strands serve as the template for the leading and lagging strands, which will be created as DNA polymerase matches complementary nucleotides to the templates; the templates may be properly referred to as the leading strand template and the lagging strand template.

DNA is read by DNA polymerase in the 3′ to 5′ direction, meaning the new strand is synthesized in the 5' to 3' direction. Since the leading and lagging strand templates are oriented in opposite directions at the replication fork, a major issue is how to achieve synthesis of new lagging strand DNA, whose direction of synthesis is opposite to the direction of the growing replication fork.

Leading strand
The leading strand is the strand of new DNA which is synthesized in the same direction as the growing replication fork. This sort of DNA replication is continuous.

Lagging strand
The lagging strand is the strand of new DNA whose direction of synthesis is opposite to the direction of the growing replication fork. Because of its orientation, replication of the lagging strand is more complicated as compared to that of the leading strand. As a consequence, the DNA polymerase on this strand is seen to "lag behind" the other strand.

The lagging strand is synthesized in short, separated segments. On the lagging strand template, a primase "reads" the template DNA and initiates synthesis of a short complementary RNA primer. A DNA polymerase extends the primed segments, forming Okazaki fragments. The RNA primers are then removed and replaced with DNA, and the fragments of DNA are joined by DNA ligase.

Dynamics at the replication fork

In all cases the helicase is composed of six polypeptides that wrap around only one strand of the DNA being replicated. The two polymerases are bound to the helicase hexamer. In eukaryotes the helicase wraps around the leading strand, and in prokaryotes it wraps around the lagging strand.

As helicase unwinds DNA at the replication fork, the DNA ahead is forced to rotate. This process results in a build-up of twists in the DNA ahead. This build-up creates a torsional load that would eventually stop the replication fork. Topoisomerases are enzymes that temporarily break the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix; topoisomerases (including DNA gyrase) achieve this by adding negative supercoils to the DNA helix.

Bare single-stranded DNA tends to fold back on itself forming secondary structures; these structures can interfere with the movement of DNA polymerase. To prevent this, single-strand binding proteins bind to the DNA until a second strand is synthesized, preventing secondary structure formation.

Double-stranded DNA is coiled around histones that play an important role in regulating gene expression so the replicated DNA must be coiled around histones at the same places as the original DNA. To ensure this, histone chaperones disassemble the chromatin before it is replicated and replace the histones in the correct place. Some steps in this reassembly are somewhat speculative.

Clamp proteins act as a sliding clamp on DNA, allowing the DNA polymerase to bind to its template and aid in processivity. The inner face of the clamp enables DNA to be threaded through it. Once the polymerase reaches the end of the template or detects double-stranded DNA, the sliding clamp undergoes a conformational change that releases the DNA polymerase. Clamp-loading proteins are used to initially load the clamp, recognizing the junction between template and RNA primers. :274-5

DNA replication proteins
At the replication fork, many replication enzymes assemble on the DNA into a complex molecular machine called the replisome. The following is a list of major DNA replication enzymes that participate in the replisome:

In vitro single-molecule experiments (using optical tweezers and magnetic tweezers) have found synergetic interactions between the replisome enzymes (helicase, polymerase, and Single-strand DNA-binding protein) and with the DNA replication fork enhancing DNA-unwinding and DNA-replication. These results lead to the development of kinetic models accounting for the synergetic interactions and their stability.

Replication machinery

Replication machineries consist of factors involved in DNA replication and appearing on template ssDNAs. Replication machineries include primosotors are replication enzymes; DNA polymerase, DNA helicases, DNA clamps and DNA topoisomerases, and replication proteins; e.g. single-stranded DNA binding proteins (SSB). In the replication machineries these components coordinate. In most of the bacteria, all of the factors involved in DNA replication are located on replication forks and the complexes stay on the forks during DNA replication. Replication machineries are also referred to as replisomes, or DNA replication systems. These terms are generic terms for proteins located on replication forks. In eukaryotic and some bacterial cells the replisomes are not formed.

In an alternative figure, DNA factories are similar to projectors and DNAs are like as cinematic films passing constantly into the projectors. In the replication factory model, after both DNA helicases for leading strands and lagging strands are loaded on the template DNAs, the helicases run along the DNAs into each other. The helicases remain associated for the remainder of replication process. Peter Meister et al. observed directly replication sites in budding yeast by monitoring green fluorescent protein (GFP)-tagged DNA polymerases α. They detected DNA replication of pairs of the tagged loci spaced apart symmetrically from a replication origin and found that the distance between the pairs decreased markedly by time. This finding suggests that the mechanism of DNA replication goes with DNA factories. That is, couples of replication factories are loaded on replication origins and the factories associated with each other. Also, template DNAs move into the factories, which bring extrusion of the template ssDNAs and new DNAs. Meister's finding is the first direct evidence of replication factory model. Subsequent research has shown that DNA helicases form dimers in many eukaryotic cells and bacterial replication machineries stay in single intranuclear location during DNA synthesis.

Replication Factories Disentangle Sister Chromatids. The disentanglement is essential for distributing the chromatids into daughter cells after DNA replication. Because sister chromatids after DNA replication hold each other by Cohesin rings, there is the only chance for the disentanglement in DNA replication. Fixing of replication machineries as replication factories can improve the success rate of DNA replication. If replication forks move freely in chromosomes, catenation of nuclei is aggravated and impedes mitotic segregation.

Termination
Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome. Because eukaryotes have linear chromosomes, DNA replication is unable to reach the very end of the chromosomes. Due to this problem, DNA is lost in each replication cycle from the end of the chromosome. Telomeres are regions of repetitive DNA close to the ends and help prevent loss of genes due to this shortening. Shortening of the telomeres is a normal process in somatic cells. This shortens the telomeres of the daughter DNA chromosome. As a result, cells can only divide a certain number of times before the DNA loss prevents further division. (This is known as the Hayflick limit.) Within the germ cell line, which passes DNA to the next generation, telomerase extends the repetitive sequences of the telomere region to prevent degradation. Telomerase can become mistakenly active in somatic cells, sometimes leading to cancer formation. Increased telomerase activity is one of the hallmarks of cancer.

Termination requires that the progress of the DNA replication fork must stop or be blocked. Termination at a specific locus, when it occurs, involves the interaction between two components: (1) a termination site sequence in the DNA, and (2) a protein which binds to this sequence to physically stop DNA replication. In various bacterial species, this is named the DNA replication terminus site-binding protein, or Ter protein.

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. E. coli regulates this process through the use of termination sequences that, when bound by the Tus protein, enable only one direction of replication fork to pass through. As a result, the replication forks are constrained to always meet within the termination region of the chromosome.

Eukaryotes
Within eukaryotes, DNA replication is controlled within the context of the cell cycle. As the cell grows and divides, it progresses through stages in the cell cycle; DNA replication takes place during the S phase (synthesis phase). The progress of the eukaryotic cell through the cycle is controlled by cell cycle checkpoints. Progression through checkpoints is controlled through complex interactions between various proteins, including cyclins and cyclin-dependent kinases. Unlike bacteria, eukaryotic DNA replicates in the confines of the nucleus.

The G1/S checkpoint (restriction checkpoint) regulates whether eukaryotic cells enter the process of DNA replication and subsequent division. Cells that do not proceed through this checkpoint remain in the G0 stage and do not replicate their DNA.

Once the DNA has gone through the "G1/S" test, it can only be copied once in every cell cycle. When the Mcm complex moves away from the origin, the pre-replication complex is dismantled. Because a new Mcm complex cannot be loaded at an origin until the pre-replication subunits are reactivated, one origin of replication can not be used twice in the same cell cycle.

Activation of S-Cdks in early S phase promotes the destruction or inhibition of individual pre-replication complex components, preventing immediate reassembly. S and M-Cdks continue to block pre-replication complex assembly even after S phase is complete, ensuring that assembly cannot occur again until all Cdk activity is reduced in late mitosis.

In budding yeast, inhibition of assembly is caused by Cdk-dependent phosphorylation of pre-replication complex components. At the onset of S phase, phosphorylation of Cdc6 by Cdk1 causes the binding of Cdc6 to the SCF ubiquitin protein ligase, which causes proteolytic destruction of Cdc6. Cdk-dependent phosphorylation of Mcm proteins promotes their export out of the nucleus along with Cdt1 during S phase, preventing the loading of new Mcm complexes at origins during a single cell cycle. Cdk phosphorylation of the origin replication complex also inhibits pre-replication complex assembly. The individual presence of any of these three mechanisms is sufficient to inhibit pre-replication complex assembly. However, mutations of all three proteins in the same cell does trigger reinitiation at many origins of replication within one cell cycle.

In animal cells, the protein geminin is a key inhibitor of pre-replication complex assembly. Geminin binds Cdt1, preventing its binding to the origin recognition complex. In G1, levels of geminin are kept low by the APC, which ubiquitinates geminin to target it for degradation. When geminin is destroyed, Cdt1 is released, allowing it to function in pre-replication complex assembly. At the end of G1, the APC is inactivated, allowing geminin to accumulate and bind Cdt1.

Replication of chloroplast and mitochondrial genomes occurs independently of the cell cycle, through the process of D-loop replication.

Replication focus
In vertebrate cells, replication sites concentrate into positions called replication foci. Replication sites can be detected by immunostaining daughter strands and replication enzymes and monitoring GFP-tagged replication factors. By these methods it is found that replication foci of varying size and positions appear in S phase of cell division and their number per nucleus is far smaller than the number of genomic replication forks.

P. Heun et al., (2001) tracked GFP-tagged replication foci in budding yeast cells and revealed that replication origins move constantly in G1 and S phase and the dynamics decreased significantly in S phase. Traditionally, replication sites were fixed on spatial structure of chromosomes by nuclear matrix or lamins. The Heun's results denied the traditional concepts, budding yeasts do not have lamins, and support that replication origins self-assemble and form replication foci.

By firing of replication origins, controlled spatially and temporally, the formation of replication foci is regulated. D. A. Jackson et al.(1998) revealed that neighboring origins fire simultaneously in mammalian cells. Spatial juxtaposition of replication sites brings clustering of replication forks. The clustering do rescue of stalled replication forks and favors normal progress of replication forks. Progress of replication forks is inhibited by many factors; collision with proteins or with complexes binding strongly on DNA, deficiency of dNTPs, nicks on template DNAs and so on. If replication forks get stuck and the rest of the sequences from the stuck forks are not copied, then the daughter strands get nick nick unreplicated sites. The un-replicated sites on one parent's strand hold the other strand together but not daughter strands. Therefore, the resulting sister chromatids cannot separate from each other and cannot divide into 2 daughter cells. When neighboring origins fire and a fork from one origin is stalled, fork from other origin access on an opposite direction of the stalled fork and duplicate the un-replicated sites. As other mechanism of the rescue there is application of dormant replication origins that excess origins do not fire in normal DNA replication.

Bacteria
Most bacteria do not go through a well-defined cell cycle but instead continuously copy their DNA; during rapid growth, this can result in the concurrent occurrence of multiple rounds of replication. In E. coli, the best-characterized bacteria, DNA replication is regulated through several mechanisms, including: the hemimethylation and sequestering of the origin sequence, the ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP), and the levels of protein DnaA. All these control the binding of initiator proteins to the origin sequences.

Because E. coli methylates GATC DNA sequences, DNA synthesis results in hemimethylated sequences. This hemimethylated DNA is recognized by the protein SeqA, which binds and sequesters the origin sequence; in addition, DnaA (required for initiation of replication) binds less well to hemimethylated DNA. As a result, newly replicated origins are prevented from immediately initiating another round of DNA replication.

ATP builds up when the cell is in a rich medium, triggering DNA replication once the cell has reached a specific size. ATP competes with ADP to bind to DnaA, and the DnaA-ATP complex is able to initiate replication. A certain number of DnaA proteins are also required for DNA replication — each time the origin is copied, the number of binding sites for DnaA doubles, requiring the synthesis of more DnaA to enable another initiation of replication.

In fast-growing bacteria, such as E. coli, chromosome replication takes more time than dividing the cell. The bacteria solve this by initiating a new round of replication before the previous one has been terminated. The new round of replication will form the chromosome of the cell that is born two generations after the dividing cell. This mechanism creates overlapping replication cycles.

Problems with DNA replication

There are many events that contribute to replication stress, including:
 * Misincorporation of ribonucleotides
 * Unusual DNA structures
 * Conflicts between replication and transcription
 * Insufficiency of essential replication factors
 * Common fragile sites
 * Overexpression or constitutive activation of oncogenes
 * Chromatin inaccessibility

Polymerase chain reaction
Researchers commonly replicate DNA in vitro using the polymerase chain reaction (PCR). PCR uses a pair of primers to span a target region in template DNA, and then polymerizes partner strands in each direction from these primers using a thermostable DNA polymerase. Repeating this process through multiple cycles amplifies the targeted DNA region. At the start of each cycle, the mixture of template and primers is heated, separating the newly synthesized molecule and template. Then, as the mixture cools, both of these become templates for annealing of new primers, and the polymerase extends from these. As a result, the number of copies of the target region doubles each round, increasing exponentially.

license: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/

title: DNA replication


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Dravidian

Dravidian, Dravidan, or Dravida may refer to:

Language and culture

 * Dravidian languages, a family of languages spoken mainly in South India and northeastern Sri Lanka
 * Proto-Dravidian language, a model of the common ancestor of the Dravidian languages
 * Dravidian University, a university situated in Andhra Pradesh
 * South Indian culture, modern Dravidian culture
 * Dravidian architecture, Hindu temple architecture

Geography

 * Dravida Nadu, a proposed country for the southern Dravidian languages
 * South India, the region which is called Dravida in the Indian anthem
 * Dravida Kingdom, an ancient region mentioned in the Mahabharata

Ethnicity

 * Dravidian peoples, ethnic groups primarily in South India.
 * Homo Dravida, a historically defined race, propagated also by Devaneya Pavanar
 * Adi Dravida, natives of Southern India

Religion

 * Dravidian folk religion

Others

 * Dravidan (1989 film), a 1989 Tamil film
 * Dravida Sangha

title: Dravidian


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Daisy Duck

Daisy Duck is an American cartoon character created by the Walt Disney Company. As the girlfriend of Donald Duck, she is an anthropomorphic white duck that has large eyelashes and ruffled tail feathers around her lowest region to suggest a skirt. She is often seen wearing a hair bow, blouse, and heeled shoes. Daisy was introduced in the short film Mr. Duck Steps Out (1940) and was incorporated into Donald's comic stories several months later. Carl Barks, the screenwriter and lead storyboard artist for the film, was inspired by the 1937 short, Don Donald, that featured a Latin character named Donna Duck, to revive the concept of a female counterpart for Donald.

Daisy appeared in 11 short films between 1940 and 1954, and far later in Mickey's Christmas Carol (1983) and Fantasia 2000 (1999). In these roles, Daisy was always a supporting character, with the exception of Donald's Dilemma (1947). Daisy has received considerably more screen time in television, making regular appearances in Quack Pack (1996), Mickey Mouse Works (1999–2000), House of Mouse (2001–2003), Mickey Mouse Clubhouse (2006–2016), Mickey Mouse (2013–2019), Mickey Mouse Mixed-Up Adventures (2017–2021), The Wonderful World of Mickey Mouse (2020–2023) and Mickey Mouse Funhouse (2021–present). Daisy has also appeared in several direct-to-video films such as Mickey's Once Upon a Christmas (1999), The Three Musketeers (2004), and Mickey's Twice Upon a Christmas (2004).

Daisy is a close friend of Clarabelle Cow and Clara Cluck in the comics and Minnie Mouse's best friend. Daisy usually shows a strong affinity towards Donald, although she is often characterized as being more sophisticated than him. Particularly in the comics, because of this, Daisy regularly becomes frustrated with Donald's immaturity, and on those occasions she will often go out on dates with Donald's cousin and rival Gladstone Gander instead. Daisy is the aunt of April, May, and June, three young girl ducks who bear resemblance to Huey, Dewey, and Louie.

Characterization
Since her early appearances, Daisy is attracted to Donald and devoted to him in the same way he is often devoted to her. This is most clearly seen in Donald's Dilemma as Daisy is almost to the point of suicide after Donald forgets her. Despite this, she is shown to have her boyfriend wrapped around her finger and is often shown to keep him in line whenever his anger starts to boil.

Besides her love for Donald, Daisy is also shown to be more sophisticated and intelligent than him. This causes her to frequently be frustrated with his immaturity, and their relationship occasionally has an off-again, on-again nature as a result, particularly in the comic books. In comics, when Daisy is fighting with Donald or temporarily breaks up with him, she goes on dates with Donald's cousin Gladstone Gander instead. In Cured Duck Daisy even gives Donald an ultimatum regarding his temper but later reforms in Donald's Dilemma. Daisy herself sometimes exhibits a temper, but she has much greater self-control than Donald.

In the Mouse Works/House of Mouse cartoons, she was sometimes portrayed as intrusive and overly talkative. She would invite herself in without asking and would tag along on trips where she was not wanted. In House of Mouse, Daisy was often waiting for her ″Big Break″, taking any and every opportunity to perform a number of talent acts on stage. Daisy was separated from Donald in that her quest for fame was not as prominent, and relied less on jealousy than eagerness.

Appearance
Daisy is a white duck with an orange bill and legs. She usually has indigo eyeshadow, long distinct eyelashes and ruffled feathers around her lowest region to suggest a skirt.

She is usually seen sporting a blouse with puffed short sleeves and a v-neckline. She also wears a matching bow, heeled shoes and a single bangle on her wrist. The colors of her clothes change very often, but her signature colors are usually purple and pink.

The creators of the television series Quack Pack, in keeping with their modernization theme, reworked Daisy's character into a career-oriented woman and thus gave her a different appearance to match. While keeping with the purple and pink motif, Daisy usually wore long dresses with high-heeled shoes and instead of wearing her trademark hair bow, the feathers atop her head got the same treatment as her tail feathers had before; the animators arranged them in such a manner to appear as if Daisy was sporting a more modern short hairstyle.

House of Mouse got her a blue and purple employee uniform, with a blue bow, earrings, and a long ponytail. In Mickey Mouse Clubhouse, Daisy regained her purple blouse with a purple bow and shoes. She also wears a gold bangle and has a short ponytail, similar to the longer one seen in House of Mouse.

Voice
Daisy Duck has been voiced by several different voice actors over the years, yet by far the most extensive work has been done by Tress MacNeille, who took on the role in 1999.

Clarence Nash voiced Daisy in her debut in Mr. Duck Steps Out. In the short, Nash voiced Daisy in a similar 'duck-like' voice as Donald's. Starting with Donald's Crime (1945), Ruth Clifford, best known as the voice of Minnie Mouse in the late 1940s and early 1950s, took over vocal duties on the character, giving her a more "normal" female human voice. Clifford would voice Daisy in a further four shorts between 1945 and 1948, with her last being Donald's Dream Voice (1948). For Donald's Dilemma (1947), actress Gloria Blondell voiced Daisy. Clifford returned to the role one final time in Crazy Over Daisy (1950). Vivi Janiss voiced the character in Donald's Diary (1954), while renowned voice actress June Foray (Rocky the Flying Squirrel) voiced her in her final classic shorts appearance, the educational Donald Duck short How to Have an Accident at Work (1959).

Voice actress Janet Waldo, best known as the voice of Judy Jetson, voiced Daisy in the Disneyland Records album An Adaptation of Dickens' Christmas Carol, Performed by The Walt Disney Players (1974).

In 1983, Daisy was voiced by Patricia Parris in Mickey's Christmas Carol. Tony Anselmo voiced Daisy in Down and Out with Donald Duck (1987). Daisy was then voiced by Kath Soucie throughout her first regular television series Quack Pack (1996). From 1989 to 1999, Daisy was voiced by Diane Michelle in the anthology film The Spirit of Mickey, the first season of Mickey Mouse Works, and other media and games at the time. Michelle alternated in the role with Tress MacNeille for Mickey's Once Upon a Christmas. In 1999, MacNeille took over as Daisy's full-time voice starting with the second season of Mickey Mouse Works. MacNeille has voiced Daisy in the television series House of Mouse, Mickey Mouse Clubhouse, Mickey Mouse, Mickey Mouse Mixed-Up Adventures, Legend of the Three Caballeros, DuckTales, and The Wonderful World of Mickey Mouse. MacNeille has also voiced Daisy in television specials, movies, and video games. Daisy was voiced by Russi Taylor in Fantasia 2000, although she has no lines other than a scream. In the second season of Mickey Mouse Funhouse, MacNeille was replaced by Debra Wilson as MacNeille was caught up in other projects. Wilson, the first African-American performer of Daisy, also voiced the character in the holiday special Mickey Saves Christmas.

Donna Duck
Donna Duck made her sole animated appearance in the short film Don Donald (1937), directed by Ben Sharpsteen. It was the first installment of the Donald Duck film series and was also the first time Donald was shown with a love interest. In the story, Donald travels to Mexico to court a duck who is largely a female version of himself. She is portrayed with the same feisty temperament and as such was also voiced by Clarence Nash. At the end of the story, she spitefully abandons Donald in the desert after his car breaks down.

While Donna was not reused in film after her only appearance, she became an inspiration for the creation of Daisy. Donna appeared in early British Disney comics and was introduced in the American comic strip in 1951, as Daisy's unwitting rival for Donald's affections.

Other Disney characters, such as Goofy, were introduced under various names (Dippy Dawg) and appearances, leading some historians to conclude that Donna and Daisy are the same character. However, in these other instances, changes usually developed over time, during which the character remained in use. Donna, on the other hand, appeared only once, and it was several years before a new female love interest for Donald was designed. There were many significant differences between the two characters all-at-once, in personality, nationality, name and attire.

According to The Encyclopedia of Animated Disney Shorts and the Big Cartoon DataBase, Don Donald is considered Daisy's debut. Don Donald is included on the Disney-produced DVD Best Pals: Donald and Daisy. In 1999, The Walt Disney Company released a collector's pin as part of their Countdown to the Millennium pin series, which reads "Daisy Duck debuts as Donna Duck 1937". Under current US copyright law, Daisy Duck is due to enter the public domain in between 2032 and 2035. However, this will only apply (at first) to the character as she is depicted as Donna Duck which was published in 1937, which will enter US public domain in 2032 with her later persona debut in 1940, which will enter US public domain in 2035. Versions of her with other later developments may persist under copyright until the works they were introduced in enter the public domain themselves.

History
Daisy debuted in theatrical animation and has appeared in a total of 15 films. She appeared in 12 Donald Duck short films. These are, in order of release, Mr. Duck Steps Out (1940), Donald's Crime (1945), Cured Duck (1945), Donald's Double Trouble (1946), Dumb Bell of the Yukon, Sleepy Time Donald (1947), Donald's Dilemma, Donald's Dream Voice (1948), Crazy Over Daisy (1950), Donald's Diary (1954) & How to Have an Accident at Work (1959) as Donald's unnamed wife. She also made a brief cameo in the Mickey Mouse short film The Nifty Nineties (1941). After the classic shorts era, Daisy appeared in Mickey's Christmas Carol (1983) and Fantasia 2000 (1999) with another cameo in Who Framed Roger Rabbit (1988).

First appearance
Daisy Duck in her familiar name and design first appeared in Mr. Duck Steps Out (June 7, 1940). The short was directed by Jack King and scripted by Carl Barks. There Donald visits the house of his new romantic interest for their first known date. At first, Daisy acts shy and has her back turned to her visitor. But Donald soon notices her tail-feathers taking the form of a hand and signaling for him to come closer. But their time alone is soon interrupted by Huey, Dewey, and Louie who have followed their uncle and clearly compete with him for the attention of Daisy. Uncle and nephews take turns dancing the jitterbug with her while trying to get rid of each other. In their final effort, the three younger Ducks feed their uncle maize (corn) in the process of becoming popcorn. The process is completed within Donald himself who continues to move spastically around the house while maintaining the appearance of dancing. The short ends with an impressed Daisy showering her new boyfriend with kisses. Like her precursor, she was initially voiced by Clarence Nash, but later had a more ladylike voice.

The short stands out among other Donald shorts of the period for its use of modern music and surreal situations throughout.

Disney shorts: 1941–1947
One year following her introduction in Mr. Duck Steps Out, Daisy, along with Donald and the nephews, made a brief cameo in the Mickey Mouse short The Nifty Nineties, cementing her position as a recurring character.

Daisy's speaking role again came 4 years later in Donald's Crime. While Daisy has a relatively small role in the film, her date with Donald is central to the plot and shows Donald's infatuation for her. Finding himself broke before the date; Donald steals money from his nephews, but afterward feels guilty. Donald imagines what Daisy might think of him knowing he stole money, and this leads him to reform in the end. Daisy was voiced in the film by actress Gloria Blondell, marking the first time Daisy had a "normal". The film also marked the first time Daisy appeared in an Academy Award nominated film (Best Animated Short).

Later that same year Daisy appeared again in Cured Duck (October 26, 1945). The short starts simply enough. Donald visits Daisy at her house. She asks him to open a window. He keeps trying to pull it open and eventually goes into a rage. By the time Daisy returns to the room, Donald has wrecked it. She demonstrates that the locking mechanism was on and criticizes his temper. She refuses to date Donald again until he learns to manage his anger. She claims Donald does not see her losing her own temper. Donald agrees to her terms and follows the surreal method of mail ordering an "insult machine", a device constantly hurling verbal and physical insults at him. He endures the whole process until feeling able to stay calm throughout it. He visits Daisy again and this time calmly opens the window. But when Daisy shows her boyfriend her new hat, his reaction is uncontrollable laughter. Daisy goes into a rage of her own and the short ends by pointing out that Donald is not the only Duck in need of anger management training. There is a continuation regarding her temper in the Mickey Mouse Works short "Donald's Dinner Date" where she and Donald have a date in a restaurant wherein they both end up with a bad temper thanks to Goofy.

Their relationship problems were also focused on in Donald's Double Trouble (June 28, 1946). This time Daisy criticizes his poor command of the English language and his less-than-refined manners. Unwilling to lose Daisy, Donald has to find an answer to the problem. But his solution involves his own look-alike who happens to have all the desired qualities. His unnamed look-alike happens to be unemployed at the moment and agrees to this plan. Donald provides the money for his dates with Daisy but soon comes to realize the look-alike serves as a rival suitor. The rest of the short focuses on his increasing jealousy and efforts to replace the look-alike during the next date. A failed attempt at a tunnel of love results in the two male Ducks exiting the tunnel in each other's hands by mistake. Daisy walks out completely drenched. She jumps up and down and sounds like a record played too fast as Donald and his look-alike run away.

In Dumb Bell of the Yukon, Daisy is the motivation behind Donald's hunting trip after he reads a letter from her saying she likes fur coats. Daisy briefly appears in a non-speaking role in Donald's daydream, imagining how pleased she will be.

Her next appearance in Sleepy Time Donald (May 9, 1947) involved Daisy attempting to rescue a sleepwalking Donald from wandering into danger. Donald is loose in an urban environment and the humor results from the problems Daisy herself suffers while trying to keep him safe.

First starring role
Daisy was the protagonist of Donald's Dilemma (July 11, 1947). In the short, Donald and Daisy are out on a date when a flower pot falls on his head. He regains consciousness soon enough but with some marked differences. Both his speaking and singing voices have been improved to the point of being able to enter a new career as a professional singer. He also acts more refined than usual. Most importantly Donald suffers from partial amnesia and has no memory of Daisy. Donald goes on becoming a well-known crooner and his rendition of When You Wish upon a Star becomes a hit. He is surrounded by female fans in his every step. Meanwhile, Daisy cannot even approach her former lover and her loss results in a number of psychological symptoms. Various scenes feature her suffering from anorexia, insomnia, and self-described insanity. An often censored scene features her losing her will to live and contemplating various methods of suicide. She narrates her story to a psychologist who determines that Donald would regain his memory with another flower pot falling on his head but warns that his improved voice may also be lost along with his singing career. He offers Daisy a dilemma. Either the world has its singer, but Daisy loses him, or Daisy regains her Donald, but the world loses him. Posed with the question "her or the world", Daisy answers with a resounding and possessive scream of "Me, Me, Me". Soon Donald has returned to his old self and has forgotten about his career. His fans forget about him. But Daisy has regained her lover. This is considered a darkly humorous look at their relationship.

Final Donald Duck shorts: 1948–1954
Daisy also appears in Donald's Dream Voice (1948), where she encourages Donald to have faith in himself.

1950's Crazy Over Daisy features Donald going to Daisy's house for a date, and getting distracted by a fight with Chip 'n Dale. The short introduced Daisy's theme song "Crazy Over Daisy", and in later appearances, Donald can be heard whistling the tune, such as in "Out on a Limb" and "Donald the Dude Duck".

Daisy's final appearance in the Golden Age of American animation was in Donald's Diary (1954). There she played the role of a beautiful lady who manages to start a long-term relationship with Donald. But after having a nightmare about the anxieties that would come from married life, Donald runs out on her and joins the French Foreign Legion. Several scenes of the short imply that Daisy has had several previous relationships with men. Donald carves their names on a tree. Not noticing than the opposing side of the tree features her name alongside that of several other boyfriends. The marriage scene in Donald's dream featured a group of sailors waving goodbye to Daisy and mourning the loss of their apparent lover. The story bore little continuity with the "real" Donald and Daisy as Huey, Dewey, and Louie appeared as Daisy's younger brothers. It was the only time in which Daisy's parents are seen.

Later theatrical appearances
In 1959, Daisy made a cameo in Donald in Mathmagic Land. When the Spirit finds Donald's mind to be too cluttered with "Antiquated Ideas", "Bungling", "False Concepts", "Superstitions", and "Confusion", there is a picture of her in the background that is signed "Love, Daisy".

Daisy appeared in Mickey's Christmas Carol in 1983, playing the character Isabelle, the neglected love interest of a young Ebenezer Scrooge, played by Scrooge McDuck. The film was Daisy's first theatrical appearance in almost 30 years and was also the first time she appeared apart from Donald, although the nature of the film was that of Disney characters "playing" other characters and was not part of any story continuity. Daisy was voiced by Patricia Parris in the film.

In 1988, Daisy made a cameo appearance in the finale of Who Framed Roger Rabbit along with many other Disney characters.

Daisy's most recent theatrical appearance was Fantasia 2000, released in late 1999. Like the original Fantasia, the film constituted various musical segments. Donald and Daisy appeared in non-speaking roles for the seventh of eight segments, set to the Pomp and Circumstance marches. The segment is a retelling of Noah's Ark with the ducks acting as Noah's assistants. Donald and Daisy become separated in the chaos of the flood and each presumes the other to have drowned until they discover each other towards the near end afterwards. Daisy kisses Donald in happiness and joy when they are reunited and the duck couple walk out of the ark hand-in-hand admiring their new home.

Non-theatrical appearances
Daisy appeared in the direct-to-video films Mickey's Once Upon a Christmas, Mickey's Twice Upon a Christmas, and Mickey, Donald, Goofy: The Three Musketeers.

In comics
According to the unofficial timeline of Don Rosa, Daisy was born in 1920. According to Rosa, Daisy is Donald's sister-in-law – Daisy's brother had married Donald's twin sister, Della Duck, and together, the two became the parents of Huey, Dewey, and Louie Duck. This is his explanation of why the triplets tend to call her "Aunt Daisy" while no such courtesy is given to Gladstone Gander for example. Don Rosa has said that he considers Donald and Daisy to be nonrelated and that Duck simply is the Duckburg universe equal to Smith, being a common surname.

Donna Duck served as a precursor for Daisy in both animation and comics. She first appeared in a one-page illustration titled "Don Donald" and published in Good Housekeeping #3701 (January 1937). The page was illustrated by Thomas "Tom" Wood (1870s – October 4, 1940) who was head of the Walt Disney Studios' publicity department from 1933 until his death. She made a brief appearance in the "Donald and Donna" comic strip published in Mickey Mouse Weekly from May 15 to August 21, 1937. The Weekly was a United Kingdom publication and the strip was illustrated at the time by William A. Ward.

Daisy made her first comics appearance on November 4, 1940. She was introduced as the new neighbor of Donald and his potential love interest. The Donald Duck comic strip was at the time scripted by Bob Karp and illustrated by Al Taliaferro. She was seemingly soft-spoken but had a fiery temper and Donald often found himself a victim to her rage. For example, one strip had Daisy waiting for Donald to carve their names and their love for each other on a tree, only to discover the male Duck had carved "Daisy loves Donald" with her name hardly visible and his name in prominent bold letters, resulting in her breaking her umbrella on his head and dismissing him as a "conceited little pup".

Her first original comic book appearance was a cameo in the story "The Mighty Trapper" by Carl Barks, first published in Walt Disney's Comics and Stories No. 36 (September 1943), wherein Huey, Dewey, and Louie ask her to lend them an old fur coat. Barks did not use the character again until "Donald Tames His Temper" (January 1946) when Daisy demands that Donald learns to manage his anger as a New Year's resolution. Donald has to agree but points early on that Daisy herself has the temper of a "wild-eyed wildcat".

Her next appearance by Barks in "Biceps Blues" (June 1946) introduced a key concept to their relationship. When Daisy seems impressed by a certain type of male, Donald is forced to emulate that type, no matter how unsuited Donald is for emulating it successfully. In this early case, Daisy envies her "old school chum" Susy Swan for dating a notable weightlifter. Donald at first protests that she seems too impressed by a "gorilla" just because the "muscle-bound buffalo" can lift 300 pounds. But when Daisy simply ignores him and daydreams about dating Hercules, Donald decides to start weightlifting. The rest of the story focuses on his ineptitude at exercising and the eventual efforts of Huey, Dewey, and Louie to cheer him up by various tricks pointing to Donald becoming stronger. But when Donald arranges a demonstration for Daisy, Susy, and her boyfriend, their tricks are not able to save him from ridicule. Daisy then chases Donald in anger (Donald, in turn, chases Huey, Dewey, and Louie in anger) while Susy boasts about her luck in men to her weightlifter boyfriend, who simply grunts and nods and fails to understand her words. Daisy failed to see that Susy's boyfriend is strong but otherwise not too gifted, whereas Donald is one who would go great lengths for her.

Daisy continued to make frequent appearances in stories by Barks but the next important one for her development was "Wintertime Wager" (January 1948). There she first attempts to act as the voice of reason between competing cousins Donald Duck and Gladstone Gander and in fact manages to prevent Donald losing his house to Gladstone because of a wager. This story established that both of them wanted to be in her good graces. Their next joined meeting in "Gladstone Returns" (August 1948) has Donald and Gladstone competing in raising enough money for her charity effort.

Their rivalry increased when "Donald's Love Letters" (December 1949) revealed that both cousins were romantically interested in Daisy. From then on many stories by both Barks and others would develop around this love triangle. Daisy in turns dates both of them but this fact does not prevent the two competing suitors from attempting to earn more of her affection or trying to embarrass each other in front of her. Daisy can be counted on to be making regular appearances alongside either of them for several years to come. Often it would appear as if Gladstone had the upper hand in winning Daisy due to his luck, only to find fate thwarts his plans, such as a contest where the man who hunts the most turkeys gets to have dinner with Daisy, who has won a beauty contest. Gladstone wins the turkey hunt but finds himself having dinner with an ugly woman who is the runner-up queen, as Daisy is incapacitated, and Donald is the one nursing her.

Similarly, Daisy's precursor Donna and Daisy herself were featured together as rivals for Donald's affection in a newspaper strip published on August 7, 1951. In her last appearance, on August 11, 1951, Donna had a fiancé, a caricature of Disney cartoonist Manuel Gonzales, establishing a distinction between her character and Daisy.

In the comics, Daisy is also a member of a local gossip group called the "Chit-Chat Society", which plays bridge and sponsors charity fund-raisers. The core membership includes Clarabelle Cow and Clara Cluck, though occasionally some other unnamed characters appear.

In later years, Carl Barks 'modernized' Daisy in two stories: 'The not-so-ancient mariner' and 'Hall of the mermaid queen'. In the first story, Daisy is wearing a lot of different wigs and outfits. Gladstone Gander is also seen wearing a wig and a new wardrobe in the story. In the second story, Daisy has short, curly hair and a bow that is much smaller than usual.

In the 1950s, Disney launched a series of stories titled "Daisy Duck's Diary", where Daisy was given more of a leading role. This series, originally by such cartoonists as Dick Moores, Jack Bradbury, Tony Strobl and Carl Barks, have continued to the present day in Italy, Denmark and the Netherlands.

Since 1999 Daisy, like Donald Duck has her own magazine in the Netherlands. She had one in Brazil between 1986 and 1997, and a short-lived series in 2004 with republications of old stories.

Super Daisy
Since the early 1970s, Daisy has been featured as a superhero crime fighter in Italian Disney comics. Daisy's alter ego as Super Daisy (Paperinika in Italian) was designed by writer Guido Martina and artist Giorgio Cavazzano as a female counterpart to the "Duck Avenger" ("Paperinik" in Italian). While Donald's superhero persona was originally created to place Donald into situations where he was finally a "winner" (versus his usual portrayal as a "loser"), when Super Daisy appeared in the same story as the Duck Avenger, she then became the "winner" and Donald was once more relegated to the role of "loser". This upset some children, who complained to the comics' editors, which resulted in the Italian comics ceasing to depict Daisy as a superhero, although Super Daisy continued to be featured in the Disney comics in Brazil.

As Super Daisy, Daisy has no superpowers but instead uses devices created by high society fashion designer Genialina Edy Son. Genialina personally designed Daisy's costume, as well as supplying her with crime-fighting gear such as sleeping pills and a James Bond-esque sports car. Frequently, Super Daisy both fights alongside and against the Duck Avenger. In the Brazilian stories, Super Daisy often teams up with other Disney comic superheroes, such as Super Goof (Goofy), Super Gilly ("Gilbert"), and the Red Bat (Fethry Duck).

While the Duck Avenger's main goal is enforcing justice in Duckburg, and proving himself better than Donald's usual, unlucky self, Super Daisy acts mostly on an extreme, somewhat warped form of feminism, donning her alternate identity to prove that women are better than men at whatever they do, openly antagonizing the Duck Avenger to prove her point. Later stories, such as the "Hero Club" inspired Italian story "Ultraheroes", show Super Daisy and the Duck Avenger at the center of a weird love triangle: Super Daisy, despite their bickering eventually warms to the Duck Avenger, feeling drawn to his righteous persona. They both feel unable to pursue their relationship, as they feel themselves cheating their non-superhero selves, as they do not realize each others' identity as companions in everyday life.

Daisy and The Mysteries of Paris
Set in Revolution-era France, Daisy and The Mysteries of Paris (French: Daisy et les mystères de Paris) is an eight-issue comic series featuring Daisy Duck as the best friend and lady in waiting of Queen Marie Ducklette. Secretly, Daisy is a mysterious masked swordswoman called The Rose of Paris, who helps the poor and those in need, as well as preventing attempts to steal the queen's throne. She must also protect her friends Donald Duck and Ludwig Von Drake from the plots of The Ignobles.

The first three issues were illustrated by Carlo Cid Lauro, and currently the series is published by Panini Italia.

The series consists of eight stories released in four issues:
 * Episode 1: "The Sapphires of the Tsar"
 * Episode 2: "The Enigma of the Seal"
 * Episode 3: "The Stagecoach to Calais"
 * Episode 4: "The Revenge of the Thistle"
 * Episode 5: "The Secret of Marie Ducklette"
 * Episode 6: "The Return of the Scarlet Swordsman"
 * Episode 7: "Escape From The Bastille"
 * Episode 8: "Adieu, Rose of Paris!"

Name in other languages

 * زيزي
 * Дейзи Дък
 * Vlatka Patka
 * Andersine And
 * Katrien Duck
 * Dúnhild Dunna
 * Iines Ankka
 * Νταίζη Ντακ
 * דייזי דאק
 * Dézi Kacsa
 * Desi Bebek
 * Andrésína Önd
 * Paperina
 * デイジーダック
 * 데이지 덕
 * Dolly Duck
 * Kaczka Daisy
 * Margarida
 * Дейзи Дак
 * ଡେ଼ସି ବତକ
 * Pata Daisy
 * Kajsa Anka
 * Deyzi
 * Пата Патка
 * Vịt Daisy
 * Vịt Daisy

Disney parks
At the Walt Disney Parks and Resorts and on the Disney Cruise Line ships, Daisy is a character for meet-and-greets, parades, and shows. Her semi-elusiveness has made her extra popular to an extent, adding to the fact that Daisy is a member of the Sensational Six, therefore making Daisy merchandise even more appealing to collectors. After Disney World expanded Fantasyland in 2012, Daisy became available for meet-and-greets at Pete's Silly Sideshow. At Epcot, she appears at the main entrance. She has also appeared in restaurants such as the Tusker House and Minnie's Springtime Diner at Hollywood & Vine.

Daisy appears in an MMORPG game called Toontown Online, based on the theme parks, where she walks around Daisy Gardens leaving comments about passing toons.

Television
In the 1996 television series Quack Pack, Daisy was presented as a much more liberated (and patient) woman than in her previous appearances, where she was employed as a television station reporter, with Donald as her cameraman. The couple also seem to have a better and steadier relationship compared to the other series. In Quack Pack, Daisy had a pet iguana named Knuckles.

Daisy also appeared in the later television series Mickey Mouse Works and House of Mouse as a regular character. She is also part of the main characters in Mickey Mouse Clubhouse and its spin-offs Minnie's Bow-Toons, Mickey Mouse Mixed-Up Adventures and Mickey Mouse Funhouse.

Daisy made her first appearance in the 2017 DuckTales series in the season 3 episode "Louie's Eleven", with MacNeille once again providing her voice. In this version, Daisy and Donald meet for the first time, and her appearance is similar to the theatrical short, Donald's Diary (1954). She is depicted as having a temper like Donald and is an assistant to Duckburg trendsetter, Emma Glamour. After Donald attempts to infiltrate one of Glamour's parties to help his band, the Three Caballeros, he and Daisy end up trapped in an elevator and develop a mutual attraction to each other. As of the episode "New Gods on the Block!", they became a couple.

Video games
In the Kingdom Hearts video game series, Daisy Duck (デイジーダック) appears as a countess in Disney Castle. In Kingdom Hearts II, she scolds Donald for being gone too long. She makes a cameo appearance in Kingdom Hearts Birth By Sleep and Kingdom Hearts III.

Daisy is a playable character in the video game Disney Think Fast and a playable race driver in the Nintendo 64 and Game Boy Color racing game Mickey's Speedway USA. She is also a playable character in Disney Golf for the PlayStation 2 and in Disney's Party for the GameCube and Game Boy Advance.

For the Wii: In Epic Mickey, a robot version of Daisy appears in the game, and in Dance Dance Revolution Disney Grooves, Daisy appears as one of the random backup dancers.

Daisy runs the Daisy Gardens neighborhood in Disney's Toontown Online.

title: Daisy Duck


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Dot-com bubble

The dot-com bubble (or dot-com boom) was a stock market bubble that ballooned during the late-1990s and peaked on Friday, March 10, 2000. This period of market growth coincided with the widespread adoption of the World Wide Web and the Internet, resulting in a dispensation of available venture capital and the rapid growth of valuations in new dot-com startups.

Between 1995 and its peak in March 2000, investments in the NASDAQ composite stock market index rose by 800%, only to fall to 78% from its peak by October 2002, giving up all its gains during the bubble.

During the dot-com crash, many online shopping companies, notably Pets.com, Webvan, and Boo.com, as well as several communication companies, such as Worldcom, NorthPoint Communications, and Global Crossing, failed and shut down. Others, like Lastminute.com, MP3.com and PeopleSound remained through its sale and buyers acquisition. Larger companies like Amazon and Cisco Systems lost large portions of their market capitalization, with Cisco losing 80% of its stock value.

Background
Historically, the dot-com boom can be seen as similar to a number of other technology-inspired booms of the past, including railroads in the 1840s, automobiles in the early 20th century, radio in the 1920s, television in the 1940s, transistor electronics in the 1950s, computer time-sharing in the 1960s, and home computers and biotechnology in the 1980s.

Overview
Low interest rates in 1998–99 facilitated an increase in start-up companies.

In 2000, the dot-com bubble burst, and many dot-com startups went out of business after burning through their venture capital and failing to become profitable. However, many others, particularly online retailers like eBay and Amazon, blossomed and became highly profitable. More conventional retailers found online merchandising to be a profitable additional source of revenue. While some online entertainment and news outlets failed when their seed capital ran out, others persisted and eventually became economically self-sufficient. Traditional media outlets (newspaper publishers, broadcasters and cablecasters in particular) also found the Web to be a useful and profitable additional channel for content distribution, and an additional means to generate advertising revenue. The sites that survived and eventually prospered after the bubble burst had two things in common: a sound business plan, and a niche in the marketplace that was, if not unique, particularly well-defined and well-served.

In the aftermath of the dot-com bubble, telecommunications companies had a great deal of overcapacity as many Internet business clients went bust. That, plus ongoing investment in local cell infrastructure kept connectivity charges low, and helped to make high-speed Internet connectivity more affordable. During this time, a handful of companies found success developing business models that helped make the World Wide Web a more compelling experience. These include airline booking sites, Google's search engine and its profitable approach to keyword-based advertising, as well as eBay's auction site and Amazon.com's online department store. The low price of reaching millions worldwide, and the possibility of selling to or hearing from those people at the same moment when they were reached, promised to overturn established business dogma in advertising, mail-order sales, customer relationship management, and many more areas. The web was a new killer app—it could bring together unrelated buyers and sellers in seamless and low-cost ways. Entrepreneurs around the world developed new business models, and ran to their nearest venture capitalist. While some of the new entrepreneurs had experience in business and economics, the majority were simply people with ideas, and did not manage the capital influx prudently. Additionally, many dot-com business plans were predicated on the assumption that by using the Internet, they would bypass the distribution channels of existing businesses and therefore not have to compete with them; when the established businesses with strong existing brands developed their own Internet presence, these hopes were shattered, and the newcomers were left attempting to break into markets dominated by larger, more established businesses.

The dot-com bubble burst in March 2000, with the technology heavy NASDAQ Composite index peaking at 5,048.62 on March 10 (5,132.52 intraday), more than double its value just a year before. By 2001, the bubble's deflation was running full speed. A majority of the dot-coms had ceased trading, after having burnt through their venture capital and IPO capital, often without ever making a profit. But despite this, the Internet continues to grow, driven by commerce, ever greater amounts of online information, knowledge, social networking and access by mobile devices.

Prelude to the bubble
The 1993 release of Mosaic and subsequent web browsers during the following years gave computer users access to the World Wide Web, popularizing use of the Internet. Internet use increased as a result of the reduction of the "digital divide" and advances in connectivity, uses of the Internet, and computer education. Between 1990 and 1997, the percentage of households in the United States owning computers increased from 15% to 35% as computer ownership progressed from a luxury to a necessity. This marked the shift to the Information Age, an economy based on information technology, and many new companies were founded.

At the same time, a decline in interest rates increased the availability of capital. The Taxpayer Relief Act of 1997, which lowered the top marginal capital gains tax in the United States, also made people more willing to make more speculative investments. Alan Greenspan, then-Chair of the Federal Reserve, allegedly fueled investments in the stock market by putting a positive spin on stock valuations. The Telecommunications Act of 1996 was expected to result in many new technologies from which many people wanted to profit.

The bubble
As a result of these factors, many investors were eager to invest, at any valuation, in any dot-com company, especially if it had one of the Internet-related prefixes or a ".com" suffix in its name. Venture capital was easy to raise. Investment banks, which profited significantly from initial public offerings (IPO), fueled speculation and encouraged investment in technology. A combination of rapidly increasing stock prices in the quaternary sector of the economy and confidence that the companies would turn future profits created an environment in which many investors were willing to overlook traditional metrics, such as the price–earnings ratio, and base confidence on technological advancements, leading to a stock market bubble. Between 1995 and 2000, the Nasdaq Composite stock market index rose 400%. It reached a price–earnings ratio of 200, dwarfing the peak price–earnings ratio of 80 for the Japanese Nikkei 225 during the Japanese asset price bubble of 1991. In 1999, shares of Qualcomm rose in value by 2,619%, 12 other large-cap stocks each rose over 1,000% in value, and seven additional large-cap stocks each rose over 900% in value. Even though the Nasdaq Composite rose 85.6% and the S&P 500 rose 19.5% in 1999, more stocks fell in value than rose in value as investors sold stocks in slower growing companies to invest in Internet stocks.

An unprecedented amount of personal investing occurred during the boom and stories of people quitting their jobs to trade on the financial market were common. The news media took advantage of the public's desire to invest in the stock market; an article in The Wall Street Journal suggested that investors "re-think" the "quaint idea" of profits, and CNBC reported on the stock market with the same level of suspense as many networks provided to the broadcasting of sports events.

At the height of the boom, it was possible for a promising dot-com company to become a public company via an IPO and raise a substantial amount of money even if it had never made a profit—or, in some cases, realized any material revenue. People who received employee stock options became instant paper millionaires when their companies executed IPOs; however, most employees were barred from selling shares immediately due to lock-up periods. The most successful entrepreneurs, such as Mark Cuban, sold their shares or entered into hedges to protect their gains. Sir John Templeton successfully shorted many dot-com stocks at the peak of the bubble during what he called "temporary insanity" and a "once-in-a-lifetime opportunity". He shorted stocks just before the expiration of lockup periods ending six months after initial public offerings, correctly anticipating many dot-com company executives would sell shares as soon as possible, and that large-scale selling would force down share prices.

Spending tendencies of dot-com companies
Most dot-com companies incurred net operating losses as they spent heavily on advertising and promotions to harness network effects to build market share or mind share as fast as possible, using the mottos "get big fast" and "get large or get lost". These companies offered their services or products for free or at a discount with the expectation that they could build enough brand awareness to charge profitable rates for their services in the future.

The "growth over profits" mentality and the aura of "new economy" invincibility led some companies to engage in lavish spending on elaborate business facilities and luxury vacations for employees. Upon the launch of a new product or website, a company would organize an expensive event called a dot-com party.

Bubble in telecom
In the five years after the American Telecommunications Act of 1996 went into effect, telecommunications equipment companies invested more than $500 billion, mostly financed with debt, into laying fiber optic cable, adding new switches, and building wireless networks. In many areas, such as the Dulles Technology Corridor in Virginia, governments funded technology infrastructure and created favorable business and tax law to encourage companies to expand. The growth in capacity vastly outstripped the growth in demand. Spectrum auctions for 3G in the United Kingdom in April 2000, led by Chancellor of the Exchequer Gordon Brown, raised £22.5 billion. In Germany, in August 2000, the auctions raised £30 billion. A 3G spectrum auction in the United States in 1999 had to be re-run when the winners defaulted on their bids of $4 billion. The re-auction netted 10% of the original sales prices. When financing became hard to find as the bubble burst, the high debt ratios of these companies led to bankruptcy. Bond investors recovered just over 20% of their investments. However, several telecom executives sold stock before the crash including Philip Anschutz, who reaped $1.9 billion, Joseph Nacchio, who reaped $248 million, and Gary Winnick, who sold $748 million worth of shares.

Bursting the bubble

Nearing the turn of the 2000s, spending on technology was volatile as companies prepared for the Year 2000 problem. There were concerns that computer systems would have trouble changing their clock and calendar systems from 1999 to 2000 which might trigger wider social or economic problems, but there was virtually no impact or disruption due to adequate preparation. Spending on marketing also reached new heights for the sector: Two dot-com companies purchased ad spots for Super Bowl XXXIII, and 17 dot-com companies bought ad spots the following year for Super Bowl XXXIV.

On January 10, 2000, America Online, led by Steve Case and Ted Leonsis, announced a merger with Time Warner, led by Gerald M. Levin. The merger was the largest to date and was questioned by many analysts. Then, on January 30, 2000, 12 ads of the 61 ads for Super Bowl XXXIV were purchased by dot-coms (sources state ranges from 12 up to 19 companies depending on the definition of dot-com company). At that time, the cost for a 30-second commercial was between $1.9 million and $2.2 million.

Meanwhile, Alan Greenspan, then Chair of the Federal Reserve, raised interest rates several times; these actions were believed by many to have caused the bursting of the dot-com bubble. According to Paul Krugman, however, "he didn't raise interest rates to curb the market's enthusiasm; he didn't even seek to impose margin requirements on stock market investors. Instead, [it is alleged] he waited until the bubble burst, as it did in 2000, then tried to clean up the mess afterward". Finance author and commentator E. Ray Canterbery agreed with Krugman's criticism.

On Friday March 10, 2000, the NASDAQ Composite stock market index peaked at 5,048.62. However, on March 13, 2000, news that Japan had once again entered a recession triggered a global sell off that disproportionately affected technology stocks. Soon after, Yahoo! and eBay ended merger talks and the Nasdaq fell 2.6%, but the S&P 500 rose 2.4% as investors shifted from strong performing technology stocks to poor performing established stocks.

On March 20, 2000, Barron's featured a cover article titled "Burning Up; Warning: Internet companies are running out of cash—fast", which predicted the imminent bankruptcy of many Internet companies. This led many people to rethink their investments. That same day, MicroStrategy announced a revenue restatement due to aggressive accounting practices. Its stock price, which had risen from $7 per share to as high as $333 per share in a year, fell $140 per share, or 62%, in a day. The next day, the Federal Reserve raised interest rates, leading to an inverted yield curve, although stocks rallied temporarily.

Tangentially to all of speculation, Judge Thomas Penfield Jackson issued his conclusions of law in the case of United States v. Microsoft Corp. (2001) and ruled that Microsoft was guilty of monopolization and tying in violation of the Sherman Antitrust Act. This led to a one-day 15% decline in the value of shares in Microsoft and a 350-point, or 8%, drop in the value of the Nasdaq. Many people saw the legal actions as bad for technology in general. That same day, Bloomberg News published a widely read article that stated: "It's time, at last, to pay attention to the numbers".

On Friday, April 14, 2000, the Nasdaq Composite index fell 9%, ending a week in which it fell 25%. Investors were forced to sell stocks ahead of Tax Day, the due date to pay taxes on gains realized in the previous year. By June 2000, dot-com companies were forced to reevaluate their spending on advertising campaigns. On November 9, 2000, Pets.com, a much-hyped company that had backing from Amazon.com, went out of business only nine months after completing its IPO. By that time, most Internet stocks had declined in value by 75% from their highs, wiping out $1.755 trillion in value. In January 2001, just three dot-com companies bought advertising spots during Super Bowl XXXV. The September 11 attacks accelerated the stock-market drop. Investor confidence was further eroded by several accounting scandals and the resulting bankruptcies, including the Enron scandal in October 2001, the WorldCom scandal in June 2002, and the Adelphia Communications Corporation scandal in July 2002.

By the end of the stock market downturn of 2002, stocks had lost $5 trillion in market capitalization since the peak. At its trough on October 9, 2002, the NASDAQ-100 had dropped to 1,114, down 78% from its peak.

Aftermath
After venture capital was no longer available, the operational mentality of executives and investors completely changed. A dot-com company's lifespan was measured by its burn rate, the rate at which it spent its existing capital. Many dot-com companies ran out of capital and went through liquidation. Supporting industries, such as advertising and shipping, scaled back their operations as demand for services fell. However, many companies were able to endure the crash; 48% of dot-com companies survived through 2004, albeit at lower valuations.

Several companies and their executives, including Bernard Ebbers, Jeffrey Skilling, and Kenneth Lay, were accused or convicted of fraud for misusing shareholders' money, and the U.S. Securities and Exchange Commission levied large fines against investment firms including Citigroup and Merrill Lynch for misleading investors.

After suffering losses, retail investors transitioned their investment portfolios to more cautious positions. Popular Internet forums that focused on high tech stocks, such as Silicon Investor, Yahoo! Finance, and The Motley Fool declined in use significantly.

Job market and office equipment glut
Layoffs of programmers resulted in a general glut in the job market. University enrollment for computer-related degrees dropped noticeably. Aeron chairs, which retailed for $1,100 each, were liquidated en masse.

Legacy
As growth in the technology sector stabilized, companies consolidated; some, such as Amazon.com, eBay, Nvidia and Google gained market share and came to dominate their respective fields. The most valuable public companies are now generally in the technology sector.

In a 2015 book, venture capitalist Fred Wilson, who funded many dot-com companies and lost 90% of his net worth when the bubble burst, said about the dot-com bubble:

"A friend of mine has a great line. He says 'Nothing important has ever been built without irrational exuberance.' Meaning that you need some of this mania to cause investors to open up their pocketbooks and finance the building of the railroads or the automobile or aerospace industry or whatever. And in this case, much of the capital invested was lost, but also much of it was invested in a very high throughput backbone for the Internet, and lots of software that works, and databases and server structure. All that stuff has allowed what we have today, which has changed all our lives... that's what all this speculative mania built."

title: Dot-com bubble