DNA replication

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DNA replication. The double helix is unwound and each strand acts as a template. Bases are
matched to synthesize the new partner strands.

DNA replication, the basis for biological inheritance, is a fundamental process that occurs in all
living organisms that copies their DNA. This process is "replication" in that each strand of the
original double-stranded DNA molecule serves as template for the reproduction of the
complementary strand. Therefore, following DNA replication, two identical DNA molecules
have been produced from a single double-stranded DNA molecule. Cellular proofreading and
error toe-checking mechanisms ensure near perfect fidelity for DNA replication.[1][2]

In a cell, DNA replication begins at specific locations in the genome, called "origins".[3]
Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. In
addition to DNA polymerase, the enzyme that synthesizes the new DNA by adding nucleotides
matched to the template strand, a number of other proteins are associated with the fork and assist
in the initiation and continuation of DNA synthesis.

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

Contents
[hide]

 1 DNA structure
 2 DNA polymerase
 3 DNA replication within the cell
o 3.1 Origins of replication
o 3.2 The replication fork
 3.2.1 Leading strand
 3.2.2 Lagging strand
 3.2.3 Dynamics at the replication fork
o 3.3 Regulation of replication
o 3.4 Termination of replication
 4 Polymerase chain reaction
 5 References
[edit] DNA structure
Main article: DNA structure
The chemical structure of 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 is a chain of four types of nucleotides
having the bases: adenine, cytosine, guanine, and thymine. A nucleotide is a mono-, di- or
triphosphate deoxyribonucleoside; that is, a deoxyribose sugar is attached to one, two or three
phosphates. Chemical interaction of these nucleotides forms phosphodiester linkages, creating
the phosphate-deoxyribose backbone of the DNA double helix with the bases pointing inward.
Nucleotides (bases) are matched between strands through hydrogen bonds to form base pairs.
Adenine pairs with thymine and cytosine pairs with guanine.

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." These terms refer to the carbon atom in
deoxyribose to which the next phosphate in the chain attaches. In addition to being
complementary, the two strands of DNA are antiparallel: they are orientated in opposite
directions. This directionality has consequences in DNA synthesis, because DNA polymerase
can only synthesize DNA in one direction by adding nucleotides to the 3' end of a DNA strand.

The pairing of bases in DNA through hydrogen bonding means that the information contained
within each strand is redundant. The nucleotides on a single strand can be used to reconstruct
nucleotides on a newly synthesized partner strand.[4]

[edit] DNA polymerase

Main article: DNA polymerase
DNA polymerases adds nucleotides to the 5' end of a strand of DNA.[5] If a mismatch is
accidentally incorporated, the polymerase is inhibited from further extension. Proofreading
removes the mismatched nucleotide and extension continues.

DNA polymerases are a family of enzymes that carry out all forms of DNA replication.[6] A
DNA polymerase can only extend an existing DNA strand paired with a template strand; it
cannot begin the synthesis of a new strand. To begin synthesis of a new strand, a short fragment
of DNA or RNA, called a primer, must be created and paired with the template strand before
DNA polymerase can synthesize new DNA.

Once a primer pairs with DNA to be replicated, DNA polymerase synthesizes 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 two of the three total phosphates attached to each
unincorporated base. (Free bases with their attached phosphate groups are called nucleoside
triphosphates.) When a nucleotide is being added to a growing DNA strand, two of the
phosphates are removed and the energy produced creates a phosphodiester (chemical) bond that
attaches the remaining phosphate to the growing chain. The energetics of this process also help
explain the directionality of synthesis - if 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.

DNA polymerases are generally extremely accurate, making less than one error for every 107
nucleotides added.[7] Even so, some DNA polymerases also have proofreading ability; they can
remove nucleotides from the end of a strand in order to correct mismatched bases. If the 5'
nucleotide needs to be removed during proofreading, the triphosphate end is lost. Hence, the
energy source that usually provides energy to add a new nucleotide is also lost.

[edit] DNA replication within the cell

Main articles: Prokaryotic DNA replication and Eukaryotic DNA replication

[edit] Origins of replication

For a cell to divide, it must first replicate its DNA.[8] This process is initiated at particular points
within the DNA, known as "origins", which are targeted by proteins that separate the two strands
and initiate DNA synthesis.[3] Origins contain DNA sequences recognized by replication initiator
proteins (e.g. dnaA in E coli' and the Origin Recognition Complex in yeast).[9] These initiator
proteins recruit other proteins to separate the two strands and initiate replication forks.

Initiator proteins recruit other proteins to separate the DNA strands at the origin, forming a
bubble. Origins tend to be "AT-rich" (rich in adenine and thymine bases) to assist this process,
because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair)—
strands rich in these nucleotides are generally easier to separate due the positive relationship
between the number of hydrogen bonds and the difficulty of breaking these bonds.[10] Once
strands are separated, RNA primers are created on the template strands. More specifically, the
leading strand receives one RNA primer per active origin of replication while the lagging strand
receives several; these several fragments of RNA primers found on the lagging strand of DNA
are called Okazaki fragments, named after their discoverer. DNA Polymerase extends the leading
strand in one continuous motion and the lagging strand in a discontinuous motion (due to the
Okazaki fragments). RNase removes the RNA fragments used to initiate replication by DNA
Polymerase, and another DNA 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.

As DNA synthesis continues, the original DNA strands continue to unwind on each side of the
bubble, forming 2 replication forks. In bacteria, which have a single origin of replication on their
circular chromosome, this process eventually 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.

[edit] The replication fork

Many enzymes are involved in the DNA replication fork.
Main article: Replication fork

The replication fork is a structure that forms within the nucleus during DNA replication. It is
created by helicases, which break the hydrogen bonds holding the two 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 polymerase matches complementary nucleotides to the templates; The templates may be
properly referred to as the leading strand template and the lagging strand template.

[edit] Leading strand

The leading strand is the template strand of the DNA double helix so that the replication fork
moves along it in the 3' to 5' direction. This allows the new strand synthesized complementary to
it to be synthesized 5' to 3' in the same direction as the movement of the replication fork.

On the leading strand, a polymerase "reads" the DNA and adds nucleotides to it continuously.
This polymerase is DNA polymerase III (DNA Pol III) in prokaryotes and presumably Pol ε[11][12]
in eukaryotes.

[edit] Lagging strand

The lagging strand is the strand of the template DNA double helix that is oriented so that the
replication fork moves along it in a 5' to 3' manner. Because of its orientation, opposite to the
working orientation of DNA polymerase III, which moves on a template in a 3' to 5' manner,
replication of the lagging strand is more complicated than that of the leading strand.

On the lagging strand, primase "reads" the DNA and adds RNA to it in short, separated
segments. In eukaryotes, primase is intrinsic to Pol α.[13] DNA polymerase III or Pol δ lengthens
the primed segments, forming Okazaki fragments. Primer removal in eukaryotes is also
performed by Pol δ.[14] In prokaryotes, DNA polymerase I "reads" the fragments, removes the
RNA using its flap endonuclease domain (RNA primers are removed by 5'-3' exonuclease
activity of polymerase I [weaver, 2005], and replaces the RNA nucleotides with DNA
nucleotides (this is necessary because RNA and DNA use slightly different kinds of nucleotides).
DNA ligase joins the fragments together.

[edit] Dynamics at the replication fork

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

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.[15] This build-up would form a
resistance that would eventually halt the progress of the replication fork. DNA topoisomerases
are enzymes that solve these physical problems in the coiling of DNA. Topoisomerase I cuts a
single backbone on the DNA, enabling the strands to swivel around each other to remove the
build-up of twists. Topoisomerase II cuts both backbones, enabling one double-stranded DNA to
pass through another, thereby removing knots and entanglements that can form within and
between DNA molecules.

Bare single-stranded DNA has a tendency to fold back upon itself and form 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.[16]

Clamp proteins form a sliding clamp around DNA, helping the DNA polymerase maintain
contact with its template and thereby assisting with 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 which
releases the DNA polymerase. Clamp-loading proteins are used to initially load the clamp,
recognizing the junction between template and RNA primers.

[edit] Regulation of replication

The cell cycle of eukaryotic cells.

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 occurs 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.[17]

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

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

Bacteria

Most bacteria do not go through a well-defined cell cycle and instead continuously copy their
DNA; during rapid growth this can result in multiple rounds of replication occurring
concurrently.[18] Within E coli, the most well-characterized bacteria, regulation of DNA
replication can be achieved through several mechanisms, including: the hemimethylation and
sequestering of the origin sequence, the ratio of ATP to ADP, and the levels of protein DnaA.
These all control the process of initiator proteins binding to the origin sequences.

Because E coli methylates GATC DNA sequences, DNA synthesis results in hemimethylated
sequences. This hemimethylated DNA is recognized by a 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.[19]

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.

[edit] Termination of replication

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 regulate
this process through the use of termination sequences which, 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.[20]

Eukaryotes initiate 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 manner. Because eukaryotes have linear chromosomes, DNA replication often
fails to synthesize to the very end of the chromosomes (telomeres), resulting in telomere
shortening. This is a normal process in somatic cells — cells are only able to 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.

[edit] Polymerase chain reaction

Main article: 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 produces amplification of 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.[21]

[edit] References
 Protein Synthesis

Legend:
Process whereby DNA encodes for the production of amino acids and proteins.

This process can be divided into two parts:

1. Transcription
Before the synthesis of a protein begins, the corresponding RNA molecule is produced by RNA
transcription. One strand of the DNA double helix is used as a template by the RNA polymerase
to synthesize a messenger RNA (mRNA). This mRNA migrates from the nucleus to the
cytoplasm. During this step, mRNA goes through different types of maturation including one
called splicing when the non-coding sequences are eliminated. The coding mRNA sequence can
be described as a unit of three nucleotides called a codon.

2. Translation
The ribosome binds to the mRNA at the start codon (AUG) that is recognized only by the
initiator tRNA. The ribosome proceeds to the elongation phase of protein synthesis. During this
stage, complexes, composed of an amino acid linked to tRNA, sequentially bind to the
appropriate codon in mRNA by forming complementary base pairs with the tRNA anticodon.
The ribosome moves from codon to codon along the mRNA. Amino acids are added one by one,
translated into polypeptidic sequences dictated by DNA and represented by mRNA. At the end, a
release factor binds to the stop codon, terminating translation and releasing the complete
polypeptide from the ribosome.

One specific amino acid can correspond to more than one codon. The genetic code is said to be
degenerate.
 

http://www.replicationfork.com/images/691px-DNA_replication.svg.png
DNA replication
From Wikipedia, the free encyclopedia
It has been suggested that Replication fork be merged into this
article or section. (Discuss)
DNA replication. The double helix is unwound and each strand acts as a
template. Bases are matched to synthesize the new partner strands.

DNA replication, the basis for biological inheritance, is a fundamental process

that occurs in all living organisms that copies their DNA. This process is
"replication" in that each strand of the original double-stranded DNA molecule
serves as template for the reproduction of the complementary strand. Therefore,
following DNA replication, two identical DNA molecules have been produced
from a single double-stranded DNA molecule. Cellular proofreading and error
toe-checking mechanisms ensure near perfectfidelity for DNA replication.[1][2]
In a cell, DNA replication begins at specific locations in the genome, called
"origins".[3] Unwinding of DNA at the origin, and synthesis of new strands, forms
a replication fork. In addition to DNA polymerase, the enzyme that synthesizes
the new DNA by adding nucleotides matched to the template strand, a number of
other proteins are associated with the fork and assist in the initiation and
continuation of DNA synthesis.
DNA replication can also be performed in vitro (outside a cell). DNA
polymerases, isolated from cells, and artificial DNA primers are used to initiate
 DNA synthesis at known sequences in a template molecule. The polymerase
chain reaction (PCR), a common laboratory technique, employs such artificial
synthesis in a cyclic manner to amplify a specific target DNA fragment from a
pool of DNA.

Contents
 [hide]

 1 DNA structure
 2 DNA polymerase
 3 DNA replication within the cell
o 3.1 Origins of replication
o 3.2 The replication fork
 3.2.1 Leading strand
 3.2.2 Lagging strand
 3.2.3 Dynamics at the
replication fork
o 3.3 Regulation of replication
o 3.4 Termination of replication
 4 Polymerase chain reaction
 5 References

[edit]DNA structure
Main article: DNA structure
The chemical structure of 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 is a
chain of four types of nucleotides having the bases: adenine, cytosine, guanine,
and thymine. A nucleotide is a mono-, di- or triphosphate deoxyribonucleoside;
that is, a deoxyribose sugar is attached to one, two or three phosphates.
Chemical interaction of these nucleotides forms phosphodiester linkages,
creating the phosphate-deoxyribose backbone of the DNA double helix with the
bases pointing inward. Nucleotides (bases) are matched between strands
through hydrogen bonds to form base pairs. Adenine pairs
with thymine and cytosinepairs with guanine.
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." These terms refer
to the carbon atom in deoxyribose to which the next phosphate in the chain
attaches. In addition to being complementary, the two strands of DNA are
antiparallel: they are orientated in opposite directions. This directionality has
consequences in DNA synthesis, because DNA polymerase can only synthesize
DNA in one direction by adding nucleotides to the 3' end of a DNA strand.
The pairing of bases in DNA through hydrogen bonding means that the
information contained within each strand is redundant. The nucleotides on a
single strand can be used to reconstruct nucleotides on a newly synthesized
partner strand.[4]

[edit]DNA polymerase
Main article: DNA polymerase
DNA polymerases adds nucleotides to the 5' end of a strand of DNA. [5] If a mismatch is
accidentally incorporated, the polymerase is inhibited from further extension. Proofreading
removes the mismatched nucleotide and extension continues.

DNA polymerases are a family of enzymes that carry out all forms of DNA

replication.[6] A DNA polymerase can only extend an existing DNA strand paired
with a template strand; it cannot begin the synthesis of a new strand. To begin
synthesis of a new strand, a short fragment of DNA or RNA, called a primer,
must be created and paired with the template strand before DNA polymerase can
synthesize new DNA.
Once a primer pairs with DNA to be replicated, DNA polymerase synthesizes 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 two of the three total phosphates attached to each
unincorporated base. (Free bases with their attached phosphate groups are
callednucleoside triphosphates.) When a nucleotide is being added to a growing
DNA strand, two of the phosphates are removed and the energy produced
creates a phosphodiester (chemical) bond that attaches the remaining phosphate
to the growing chain. The energetics of this process also help explain the
directionality of synthesis - if 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.
DNA polymerases are generally extremely accurate, making less than one error
for every 107nucleotides added.[7] Even so, some DNA polymerases also have
proofreading ability; they can remove nucleotides from the end of a strand in
order to correct mismatched bases. If the 5' nucleotide needs to be removed
during proofreading, the triphosphate end is lost. Hence, the energy source that
usually provides energy to add a new nucleotide is also lost.

[edit]DNA replication within the cell

Main articles: Prokaryotic DNA replication and Eukaryotic DNA replication
[edit]Origins of replication
For a cell to divide, it must first replicate its DNA.[8] This process is initiated at
particular points within the DNA, known as "origins", which are targeted by
proteins that separate the two strands and initiate DNA synthesis.[3] Origins
contain DNA sequences recognized by replication initiator proteins
(e.g. dnaA in E coli' and the Origin Recognition Complex in yeast).[9] These
initiator proteins recruit other proteins to separate the two strands and initiate
replication forks.
Initiator proteins recruit other proteins to separate the DNA strands at the origin,
forming a bubble. Origins tend to be "AT-rich" (rich in adenine and thymine
bases) to assist this process, because A-T base pairs have two hydrogen bonds
(rather than the three formed in a C-G pair)—strands rich in these nucleotides
are generally easier to separate due the positive relationship between the
number of hydrogen bonds and the difficulty of breaking these bonds.[10] Once
strands are separated, RNA primers are created on the template strands. More
specifically, the leading strand receives one RNA primer per active origin of
replication while the lagging strand receives several; these several fragments of
RNA primers found on the lagging strand of DNA are called Okazaki fragments,
named after their discoverer. DNA Polymerase extends the leading strand in one
continuous motion and the lagging strand in a discontinuous motion (due to the
Okazaki fragments). RNase removes the RNA fragments used to initiate
replication by DNA Polymerase, and another DNA 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.
As DNA synthesis continues, the original DNA strands continue to unwind on
each side of the bubble, forming 2 replication forks. In bacteria, which have a
single origin of replication on their circular chromosome, this process eventually
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.
[edit]The replication fork

Many enzymes are involved in the DNA replication fork.

Main article: Replication fork

The replication fork is a structure that forms within the nucleus during DNA
replication. It is created by helicases, which break the hydrogen bonds holding
the two 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
polymerase matches complementary nucleotides to the templates; The templates
may be properly referred to as the leading strand template and the lagging strand
template.
[edit]Leading strand
The leading strand is the template strand of the DNA double helix so that the
replication fork moves along it in the 3' to 5' direction. This allows the new strand
synthesized complementary to it to be synthesized 5' to 3' in the same direction
as the movement of the replication fork.
On the leading strand, a polymerase "reads" the DNA and adds nucleotides to it
continuously. This polymerase is DNA polymerase III (DNA Pol III)
in prokaryotes and presumably Pol ε[11][12] in eukaryotes.
[edit]Lagging strand
The lagging strand is the strand of the template DNA double helix that is oriented
so that the replication fork moves along it in a 5' to 3' manner. Because of its
orientation, opposite to the working orientation of DNA polymerase III, which
moves on a template in a 3' to 5' manner, replication of the lagging strand is
more complicated than that of the leading strand.
On the lagging strand, primase "reads" the DNA and adds RNA to it in short,
separated segments. In eukaryotes, primase is intrinsic to Pol α.[13] DNA
polymerase III or Pol δ lengthens the primed segments, forming Okazaki
fragments. Primer removal in eukaryotes is also performed by Pol δ.[14] In
prokaryotes, DNA polymerase I "reads" the fragments, removes the RNA using
its flap endonuclease domain (RNA primers are removed by 5'-3' exonuclease
activity of polymerase I [weaver, 2005], and replaces the RNA nucleotides with
DNA nucleotides (this is necessary because RNA and DNA use slightly different
kinds of nucleotides). DNA ligase joins the fragments together.
[edit]Dynamics at the replication fork
The assembled human DNA clamp, atrimer of the protein PCNA.

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.[15] This build-
up would form a resistance that would eventually halt the progress of the
replication fork. DNA topoisomerases are enzymes that solve these physical
problems in the coiling of DNA. Topoisomerase I cuts a single backbone on the
DNA, enabling the strands to swivel around each other to remove the build-up of
twists. Topoisomerase II cuts both backbones, enabling one double-stranded
DNA to pass through another, thereby removing knots and entanglements that
can form within and between DNA molecules.
Bare single-stranded DNA has a tendency to fold back upon itself and
form 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.[16]
Clamp proteins form a sliding clamp around DNA, helping the DNA polymerase
maintain contact with its template and thereby assisting with 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 which releases the DNA
polymerase. Clamp-loading proteins are used to initially load the clamp,
recognizing the junction between template and RNA primers.
[edit]Regulation of replication

The cell cycle of eukaryotic cells.

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 occurs 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 andcyclin-dependent
kinases.[17]
The G1/S checkpoint (or restriction checkpoint) regulates whether eukaryotic
cells enter the process of DNA replication and subsequent division. Cells which
do not proceed through this checkpoint are quiescent in the "G0" stage and do
not replicate their DNA.
Replication of chloroplast and mitochondrial genomes occurs independent of the
cell cycle, through the process of D-loop replication.
Bacteria
Most bacteria do not go through a well-defined cell cycle and instead
continuously copy their DNA; during rapid growth this can result in multiple
rounds of replication occurring concurrently.[18] Within E coli, the most well-
characterized bacteria, regulation of DNA replication can be achieved through
several mechanisms, including: the hemimethylation and sequestering of the
origin sequence, the ratio of ATP to ADP, and the levels of protein DnaA. These
all control the process of initiator proteins binding to the origin sequences.
Because E coli methylates GATC DNA sequences, DNA synthesis results in
hemimethylated sequences. This hemimethylated DNA is recognized by a
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.[19]
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.
[edit]Termination of replication
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 regulate this process through the use of termination
sequences which, 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.[20]
Eukaryotes initiate 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 manner. Because eukaryotes
have linear chromosomes, DNA replication often fails to synthesize to the very
end of the chromosomes (telomeres), resulting in telomere shortening. This is a
normal process in somatic cells — cells are only able to 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.

[edit]Polymerase chain reaction

Main article: 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 produces amplification of 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.[21]

[edit]References
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