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In molecular biology , DNA
DNA
REPLICATION is the biological process of producing two identical replicas of DNA
DNA
from one original DNA molecule. This process occurs in all living organisms and is the basis for biological inheritance . The cell possesses the distinctive property of division, which makes replication of DNA
DNA
essential.

DNA
DNA
is made up of a double helix of two complementary strands. During replication, these strands are separated. Each strand of the original DNA
DNA
molecule then serves as a template for the production of its counterpart, a process referred to as semiconservative replication . Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA
DNA
replication.

In a cell , DNA
DNA
replication begins at specific locations, or origins of replication , in the genome . Unwinding of DNA
DNA
at the origin and synthesis of new strands 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
DNA
synthesis. Most prominently, DNA
DNA
polymerase synthesizes the new strands by adding nucleotides that complement each (template) strand. DNA
DNA
replication occurs during the S-stage of interphase .

DNA
DNA
replication can also be performed in vitro (artificially, outside a cell). DNA
DNA
polymerases isolated from cells and artificial DNA primers can be used to initiate DNA
DNA
synthesis at known sequences in a template DNA
DNA
molecule. The polymerase chain reaction (PCR), a common laboratory technique, cyclically applies such artificial synthesis to amplify a specific target DNA
DNA
fragment from a pool of DNA.

CONTENTS

* 1 DNA
DNA
structures * 2 DNA
DNA
polymerase

* 3 Replication process

* 3.1 Initiation * 3.2 Elongation

* 3.3 Replication fork
Replication fork

* 3.3.1 Leading strand * 3.3.2 Lagging strand * 3.3.3 Dynamics at the replication fork

* 3.4 DNA
DNA
replication proteins * 3.5 Replication machinery * 3.6 Termination

* 4 Regulation

* 4.1 Eukaryotes

* 4.1.1 Replication focus

* 4.2 Bacteria
Bacteria

* 5 Polymerase chain reaction
Polymerase chain reaction
* 6 See also * 7 Notes * 8 References

DNA
DNA
STRUCTURES

DNA
DNA
usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix . Each single strand of DNA
DNA
is a chain of four types of nucleotides . Nucleotides
Nucleotides
in DNA
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
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
DNA
double helix with the nuclei bases pointing inward (i.e., toward the opposing strand). Nucleotides
Nucleotides
(bases) are matched between strands through hydrogen bonds to form base pairs . Adenine
Adenine
pairs with thymine (two hydrogen bonds), and guanine pairs with cytosine (stronger: three hydrogen bonds).

DNA
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
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
DNA
synthesis, because DNA polymerase can synthesize DNA
DNA
in only one direction by adding nucleotides to the 3' end of a DNA
DNA
strand.

The pairing of complementary bases in DNA
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. This allows the strands to be separated from one another. The nucleotides on a single strand can therefore be used to reconstruct nucleotides on a newly synthesized partner strand.

DNA
DNA
POLYMERASE

Main article: DNA
DNA
polymerase DNA
DNA
polymerases adds nucleotides to the 3' end of a strand of DNA. If a mismatch is accidentally incorporated, the polymerase is inhibited from further extension. Proofreading removes the mismatched nucleotide and extension continues.

DNA
DNA
polymerases are a family of enzymes that carry out all forms of DNA
DNA
replication. DNA
DNA
polymerases in general cannot initiate synthesis of new strands, but can only extend an existing DNA
DNA
or RNA
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
DNA
strand.

DNA
DNA
polymerase adds a new strand of DNA
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
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
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 phosphates 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
DNA
polymerases are highly accurate, with an intrinsic error rate of less than one mistake for every 107 nucleotides added. In addition, some DNA
DNA
polymerases also have proofreading ability; they can remove nucleotides from the end of a growing strand in order to correct mismatched bases. Finally, post-replication mismatch repair mechanisms monitor the DNA
DNA
for errors, being capable of distinguishing mismatches in the newly synthesized DNA
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
DNA
replication in a living cell was first measured as the rate of phage T4 DNA
DNA
elongation in phage-infected E. coli. During the period of exponential DNA
DNA
increase at 37 °C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA
DNA
synthesis is 1.7 per 108.

REPLICATION PROCESS

Main articles: Prokaryotic DNA
DNA
replication and Eukaryotic DNA replication

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

INITIATION

Role of initiators for initiation of DNA
DNA
replication. Formation of pre-replication complex.

For a cell to divide , it must first replicate its DNA. This process is initiated at particular points in the DNA, known as "origins ", which are targeted by initiator proteins . In E. coli this protein is DnaA ; 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. Once the origin has been located, these initiators recruit other proteins and form the pre-replication complex , which unzips the double-stranded DNA.

ELONGATION

DNA
DNA
polymerase has 5'-3' activity. All known DNA
DNA
replication systems require a free 3' hydroxyl group before synthesis can be initiated (note: the DNA
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
DNA
synthesis are recognized:

* All cellular life forms and many DNA
DNA
viruses , phages and plasmids use a primase to synthesize a short RNA
RNA
primer with a free 3' OH group which is subsequently elongated by a DNA
DNA
polymerase. * The retroelements (including retroviruses ) employ a transfer RNA that primes DNA
DNA
replication by providing a free 3′ OH that is used for elongation by the reverse transcriptase . * 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
DNA
polymerase to form a new strand. * In the single stranded DNA
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
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
DNA
polymerase to synthesize the new strand.

The first is the best known of these mechanisms and is used by the cellular organisms. In this mechanism, once the two strands are separated, primase adds RNA
RNA
primers to the template strands. The leading strand receives one RNA
RNA
primer while the lagging strand receives several. The leading strand is continuously extended from the primer by a DNA
DNA
polymerase with high processivity , while the lagging strand is extended discontinuously from each primer forming Okazaki fragments . RNase removes the primer RNA
RNA
fragments, and a low processivity DNA
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
DNA
molecule.

The primase used in this process differs significantly between bacteria and archaea /eukaryotes . Bacteria
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
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
DNA
polymerases of the A/B/Y families that are involved in DNA
DNA
replication and repair. In eukaryotic replication, the primase forms a complex with Pol α.

Multiple DNA
DNA
polymerases take on different roles in the DNA replication process. In E. coli , DNA
DNA
Pol III is the polymerase enzyme primarily responsible for DNA
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
DNA
Pol I
Pol I
is the enzyme responsible for replacing RNA primers with DNA. DNA
DNA
Pol I
Pol I
has a 5' to 3' exonuclease activity in addition to its polymerase activity, and uses its exonuclease activity to degrade the RNA
RNA
primers ahead of it as it extends the DNA
DNA
strand behind it, in a process called nick translation . Pol I
Pol I
is much less processive than Pol III because its primary function in DNA replication is to create many short DNA
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
DNA
synthesis continues, the original DNA
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

Scheme of the replication fork. a: template, b: leading strand, c: lagging strand, d: replication fork, e: primer, f: Okazaki fragments Many enzymes are involved in the DNA
DNA
replication fork.

The replication fork is a structure that forms within the nucleus during DNA
DNA
replication. It is created by helicases, which break the hydrogen bonds holding the two DNA
DNA
strands together. 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
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
DNA
IS ALWAYS 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 nascent (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 nascent DNA
DNA
which is being synthesized in the same direction as the growing replication fork. A polymerase "reads" the leading strand template and adds complementary nucleotides to the nascent leading strand on a continuous basis.

Lagging Strand

The lagging strand is the strand of nascent DNA
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
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
DNA
and initiates synthesis of a short complementary RNA
RNA
primer. A DNA polymerase extends the primed segments, forming Okazaki fragments . The RNA
RNA
primers are then removed and replaced with DNA, and the fragments of DNA
DNA
are joined together by DNA
DNA
ligase .

Dynamics At The Replication Fork

The assembled human DNA
DNA
clamp, a trimer of the protein PCNA
PCNA
.

As helicase unwinds DNA
DNA
at the replication fork, the DNA
DNA
ahead is forced to rotate. This process results in a build-up of twists in the DNA
DNA
ahead. This build-up forms a torsional resistance that would eventually halt the progress of 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
DNA
helix; topoisomerases (including DNA
DNA
gyrase ) achieve this by adding negative supercoils to the DNA
DNA
helix.

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

Clamp proteins form a sliding clamp around DNA, helping the DNA polymerase maintain contact with its template, thereby assisting with processivity. The inner face of the clamp enables DNA
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
DNA
polymerase. Clamp-loading proteins are used to initially load the clamp, recognizing the junction between template and RNA
RNA
primers. :274-5

DNA
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
DNA
replication enzymes that participate in the replisome:

ENZYME FUNCTION IN DNA
DNA
REPLICATION

DNA
DNA
Helicase
Helicase
Also known as helix destabilizing enzyme. Helicase
Helicase
separates the two strands of DNA
DNA
at the Replication Fork
Replication Fork
behind the topoisomerase.

DNA
DNA
Polymerase The enzyme responsible for catalyzing the addition of nucleotide substrates to DNA
DNA
in the 5' to 3' direction during DNA
DNA
replication. Also performs proof-reading and error correction. There exist many different types of DNA
DNA
Polymerase, (such as the DNA
DNA
Polymerase III )each of which perform different functions in different types of cells.

DNA
DNA
clamp A protein which prevents elongating DNA
DNA
polymerases from dissociating from the DNA
DNA
parent strand.

Single-Strand Binding (SSB) Proteins Bind to ss DNA
DNA
and prevent the DNA
DNA
double helix from re-annealing after DNA
DNA
helicase unwinds it, thus maintaining the strand separation, and facilitating the synthesis of the nascent strand.

Topoisomerase Relaxes the DNA
DNA
from its super-coiled nature.

DNA
DNA
Gyrase Relieves strain of unwinding by DNA
DNA
helicase; this is a specific type of topoisomerase

DNA
DNA
Ligase Re-anneals the semi-conservative strands and joins Okazaki Fragments of the lagging strand.

Primase
Primase
Provides a starting point of RNA
RNA
(or DNA) for DNA
DNA
polymerase to begin synthesis of the new DNA
DNA
strand.

Telomerase Lengthens telomeric DNA
DNA
by adding repetitive nucleotide sequences to the ends of EUKARYOTIC CHROMOSOMES . This allows germ cells and stem cells to avoid the Hayflick limit on cell division.

REPLICATION MACHINERY

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

Since replication machineries do not move relatively to template DNAs such as factories, they are called a REPLICATION FACTORY. In an alternative figure, DNA
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
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
DNA
polymerases α. They detected DNA
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
DNA
replication goes with DNA
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 nascent 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
DNA
synthesis.

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

TERMINATION

Eukaryotes initiate DNA
DNA
replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome; these are not known to be regulated in any particular way. Because eukaryotes have linear chromosomes, DNA
DNA
replication is unable to reach the very end of the chromosomes, but ends at the telomere region of repetitive DNA
DNA
close to the ends. This shortens the telomere of the daughter DNA
DNA
strand. Shortening of the telomeres is a normal process in somatic cells . As a result, cells can only divide a certain number of times before the DNA
DNA
loss prevents further division. (This is known as the Hayflick limit .) Within the germ cell line, which passes DNA
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
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
DNA
replication. In various bacterial species, this is named the DNA
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.

REGULATION

Main articles: Cell division and Cell cycle The cell cycle of eukaryotic cells.

EUKARYOTES

Within eukaryotes, DNA
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
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
DNA
replicates in the confines of the nucleus.

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

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 don't 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 stall and the remaining sequences from the stalled forks are not replicated, the daughter strands have nick obtained un-replicated 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 don't fire in normal DNA
DNA
replication.

BACTERIA

Dam methylates adenine of GATC sites after replication.

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
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
DNA
sequences, DNA
DNA
synthesis results in hemimethylated sequences. This hemimethylated DNA
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
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. See also: Plasmid
Plasmid
, Plasmid
Plasmid
copy number , and Plasmid
Plasmid
partition system

POLYMERASE CHAIN REACTION

Main article: Polymerase chain reaction
Polymerase chain reaction

Researchers commonly replicate DNA
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
DNA
polymerase . Repeating this process through multiple cycles amplifies the targeted DNA
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 .

SEE ALSO

Wikimedia Commons has media related to DNA
DNA
REPLICATION .

* Autopoiesis * Data storage device * Self-replication
Self-replication

NOTES

* ^ The energetics of this process may also help explain the directionality of synthesis—if DNA
DNA
were synthesized in the 3' to 5' direction, the energy for the process would come from the 5' end of the growing strand rather than from free nucleotides. The problem is that if the high energy triphosphates were on the growing strand and not on the free nucleotides, proof-reading by removing a mismatched terminal nucleotide would be problematic: Once a nucleotide is added, the triphosphate is lost and a single phosphate remains on the backbone between the new nucleotide and the rest of the strand. If the added nucleotide were mismatched, removal would result in a DNA
DNA
strand terminated by a monophosphate at the end of the "growing strand" rather than a high energy triphosphate. So strand would be stuck and wouldn't be able to grow anymore. In actuality, the high energy triphosphates hydrolyzed at each step originate from the free nucleotides, not the polymerized strand, so this issue does not exist.

REFERENCES

* ^ Imperfect DNA
DNA
replication results in mutations . Berg JM, Tymoczko JL, Stryer L, Clarke ND (2002). "Chapter 27: DNA
DNA
Replication, Recombination, and Repair". Biochemistry. W.H. Freeman and Company. ISBN 0-7167-3051-0 . External link in chapter= (help ) * ^ A B Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). "5 DNA
DNA
Replication, Repair, and Recombination". Molecular Biology of the Cell. Garland Science. ISBN 0-8153-3218-1 . External link in chapter= (help ) * ^ A B Berg JM, Tymoczko JL, Stryer L, Clarke ND (2002). "Chapter 27, Section 4: DNA
DNA
Replication of Both Strands Proceeds Rapidly from Specific Start Sites". Biochemistry. W.H. Freeman and Company. ISBN 0-7167-3051-0 . External link in chapter= (help ) * ^ Alberts, B., et al., Molecular Biology of the Cell, Garland Science, 4th ed., 2002, pp. 238–240 ISBN 0-8153-3218-1 * ^ Allison, Lizabeth A. Fundamental Molecular Biology. Blackwell Publishing. 2007. p.112 ISBN 978-1-4051-0379-4 * ^ Berg JM, Tymoczko JL, Stryer L, Clarke ND (2002). Biochemistry. W.H. Freeman and Company. ISBN 0-7167-3051-0 . Chapter 27, Section 2: DNA
DNA
Polymerases Require a Template
Template
and a Primer * ^ A B McCulloch, Scott D; Kunkel, Thomas A (January 2008). "The fidelity of DNA
DNA
synthesis by eukaryotic replicative and translesion synthesis polymerases" . Cell Research. 18 (1): 148–161. PMC 3639319  . PMID 18166979 . doi :10.1038/cr.2008.4 . * ^ McCarthy, David; Minner, Charles; Bernstein, Harris; Bernstein, Carol (October 1976). " DNA
DNA
elongation rates and growing point distributions of wild-type phage T4 and a DNA-delay amber mutant". Journal of Molecular Biology. 106 (4): 963–981. PMID 789903 . doi :10.1016/0022-2836(76)90346-6 . * ^ Drake JW (1970) The Molecular Basis of Mutation. Holden-Day, San Francisco ISBN 0816224501 ISBN 978-0816224500 * ^ Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). Molecular Biology of the Cell. Garland Science. ISBN 0-8153-3218-1 . Chapter 5: DNA
DNA
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* v * t * e

DNA
DNA
replication (comparing Prokaryotic to Eukaryotic )

INITIATION

Prokaryotic (initiation )

* Pre-replication complex
Pre-replication complex

* dnaC

* Cdc6

* Helicase
Helicase

* dnaA * dnaB * T7

* Primase
Primase

* dnaG

Eukaryotic (preparation in G1 phase)

* Pre-replication complex
Pre-replication complex

* Origin recognition complex

* ORC1 * ORC2
ORC2
* ORC3
ORC3
* ORC4
ORC4
* ORC5 * ORC6

* Cdc6

* Cdt1

* Minichromosome maintenance

* MCM2 * MCM3 * MCM4 * MCM5 * MCM6 * MCM7

* Licensing factor

* Autonomously replicating sequence

* Single-strand binding protein

* SSBP2 * SSBP3 * SSBP4

* RNase H

* RNASEH1 * RNASEH2A

* Helicase
Helicase
: HFM1

* Primase
Primase
: PRIM1
PRIM1
* PRIM2

BOTH

* Origin of replication /Ori /Replicon

* Replication fork
Replication fork

* Lagging and leading strands

* Okazaki fragments * Primer

REPLICATION

Prokaryotic (elongation )

* DNA
DNA
polymerase III holoenzyme

* dnaC * dnaE * dnaH * dnaN * dnaQ * dnaT * dnaX * holA * holB * holC * holD * holE *

* Replisome * DNA
DNA
ligase * DNA
DNA
clamp

* Topoisomerase

* DNA
DNA
gyrase

* Prokaryotic DNA
DNA
polymerase : DNA
DNA
polymerase I

* Klenow fragment
Klenow fragment

Eukaryotic (synthesis in S phase)

* Replication factor C

* RFC1

* Flap endonuclease

* FEN1

* Topoisomerase

* Replication protein A

* RPA1

* Eukaryotic DNA