DNA replication

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 occurring in all living organisms to copy their DNA. This process is "semiconservative" in that each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Hence, following DNA replication, two identical DNA molecules have been produced from a single double -stranded DNA molecule. Cellular proofreading and error-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.

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: adenine, cytosine, guanine, and thymine. A nucleotide is a triphosphate deoxyribonucleoside; that is, a deoxyribose sugar attached to a trip hosphate and a base. Chemical interaction of these nucleotides forms phosphodiester linkages, creating the phosphate-deoxribose backbone of the DNA double helix with the bases pointing inward. Nucleotides (bases) are matched between strands throughhydrogen 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' end" and the "5' end." These terms refer to the carbon atom in ribose to which the nex t phosphate in the chain attaches. In addition to being complementary, the two strands of DNA are antiparallel: they are oriented in opposite directions. This directionality has consequences in DNA synthesis, because DNA polymerase can only synthesize DNAin 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]

DNA polymerase

DNA polymerase 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 polymerases are a family of enzymes that carry out all forms of DNA replication.[5] 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 newnucleotides 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 aphosphodiester (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.
7 DNA polymerases are generally extremely accurate, making less than one error for every 10 [6] nucleotides added. 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.

DNA polymerase

3D structure of the DNA-binding helix-turn-helix motifs in human DNA polymerase beta A DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best-known for their role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand. The newly-polymerized molecule is complementary to the template strand and identical to the template's original partner strand. DNA polymerases use a magnesium ion for catalytic activity.
y

Function

DNA polymerase with proofreading ability DNA polymerase can add free nucleotides to only the 3 end of the newly -forming strand. This results in elongation of the new strand in a 5'-3' direction. No known DNA polymerase is able to begin a new chain (de novo). DNA polymerase can add a nucleotide onto only a preexisting 3'-OH group, and, therefore, needs a primer at which it can add the first nucleotide. Primers consist of RNA and DNA bases with the first two bases always being RNA, and are synthesized by another enzyme called primase. An enzyme known as a helicase is required to unwind DNA from a double-strand structure to a single-strand structure to facilitate replication of each strand consistent with the semiconservative model of DNA replication. Error correction is a property of some, but not all, DNA polymerases. This process corrects mistakes in newly-synthesized DNA. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA. The 3' >5' exonuclease activity of the enzyme allows the incorrect base pair to be excised (this activity is known as proofreading). Following base excision, the polymerase can re-insert the correct base and replication can continue.

Variation across species

DNA polymerases have highly-conserved structure, which means that their overall catalytic subunits vary, on a whole, very little from species to species. Conserved structures usually indicate important, irreplicable functions of the cell, the maintenance of which provides evolutionary advantages.

Some vi es also encode special DNA pol merases, such as Hepatitis B virus DNA pol merase. These may selectively replicate viral DNA through a variety of mechanisms. Retroviruses encode an unusual DNA polymerase called reverse transcriptase, which is an RNA-dependent DNA polymerase (RdDp). It polymeri es DNA from a template of RNA.

DNA pol

famili

Based on sequence homology, DNA polymerases can be further subdivided into seven different families: A, B, C, D, X, Y, and RT.

Famil A
Polymerases contain both replicative and repair polymerases. Replicative members from this family include the extensively-studied T7 DNA polymerase, as well as the eukaryotic mitochondrial DNA Polymerase . Among the repair polymerases are E. coli DNA pol I, Th mus aquaticus pol I, and Bacillus stearothermophilus pol I. These repair polymerases are involved in excision repair and processing of Okazaki fragments generated during lagging strand synthesis.

Famil B
Polymerases mostly contain replicative polymerases and include the major eukaryotic DNA polymerases , , , (see Greek letters) and also DNA polymerase . Family B also includes DNA polymerases encoded by some bacteria and bacteriophages, of which the bestcharacterized are from T4, Phi 9, and RB 9 bacteriophages. These enzymes are involved in both leading and lagging strand synthesis. A hallmark of the B family of polymerases is remarkable accuracy during replication; and many have strong 3'-5' exonuclease activity (except DNA polymerase and , which have no proofreading activity).

Famil C
Polymerases are the primary bacterial chromosomal replicative enzymes. DNA Polymerase III alpha subunit from E. coli is the catalytic subunit [1] and possesses no known nuclease activity. A separate subunit, the epsilon subunit, possesses the 3'-5' exonuclease activity used for editing during chromosomal replication. Recent research has classified Family C polymerases as a subcategory of Family X.

Famil D
Polymerases are still not very well characterized. All known examples are found in the Euryarchaeota subdomain of Archaea and are thought to be replicative polymerases.

Famil X
Contains the well-known eukaryotic polymerase pol , as well as other eukaryotic polymerases such as pol , pol , pol , and terminal deoxynucleotidyl transferase (TdT). Pol is required for short-patch base excision repair, a DNA repair pathway that is essential for repairing abasic sites. Pol and Pol are involved in non-homologous end-joining, a mechanism for rejoining DNA double-strand breaks. TdT is expressed only in lymphoid

tissue, and adds "n nucleotides" to double-strand breaks formed during V(D)J recombination to promote immunological diversity. The yeast Saccharomyces cerevisiae has only one Pol X polymerase, Pol4, which is involved in non-homologous end-joining.

Famil Y
Polymerases differ from others in having a low fidelity on undamaged templates and in their ability to replicate through damaged DNA. Members of this family are hence called translesion synthesis (TLS) polymerases. Depending on the lesion, TLS polymerases can bypass the damage in an error-free or error-prone fashion, the latter resulting in elevated mutagenesis. Xeroderma pigmentosum variant (XPV) patients for instance have mutations in the gene encoding Pol (eta), which is error-free for UV-lesions. In XPV patients, alternative error-prone polymerases, e.g., Pol (zeta) (polymerase is a B Family polymerase a complex of the catalytic subunit REV3L with Rev7, which associates with Rev1[2] ), are thought to be involved in mistakes that result in the cancer predisposition of these patients. Other members in humans are Pol (iota), Pol (kappa), and Rev1 (terminal deoxycytidyl transferase). In E.coli, two TLS polymerases, Pol IV (DINB) and PolV (UmuD'2C), are known.

Famil RT
The reverse transcriptase family contains examples from both retroviruses and eukaryotic polymerases. The eukaryotic polymerases are usually restricted to telomerases. These polymerases use an RNA template to synthesize the DNA strand.

Prokaryoti DNA polymerases

Bacteria have 5 known DNA polymerases:
y y y y y

Pol I: implicated in DNA repair; has 5'->3' (Polymerase) activity and both 3'->5' exonuclease (Proofreading) and 5'->3' exonuclease activity (RNA Primer removal). Pol II: involved in reparation of damaged DNA; has 3'->5' exonuclease activity. Pol III: the main polymerase in bacteria (elongates in DNA replication); has 3'->5' exonuclease proofreading ability. Pol I : a Y-family DNA polymerase. Pol : a Y-family DNA polymerase; participates in bypassing DNA damage.

Eukaryoti DNA polymerases

Eukaryotes have at least 15 DNA Polymerases:[3]
y

y y

Pol (synonyms are RNA primase, DNA polymerase): forms a complex with a small catalytic (PriS) and a large noncatalytic (PriL) subunit [4], with the Pri subunits acting as a primase (synthesizing an RNA primer), and then with DNA Pol elongating that primer with DNA nucleotides. After around 20 nucleotides[5] elongation is taken over by Pol (on the leading strand) and (on the lagging strand). Pol : Implicated in repairing DNA, in base excision repair and gap-filling synthesis. Pol : Replicates and repairs mitochondrial DNA and has proofreading 3'->5' exonuclease activity.

y y

Pol : Highly processive and has proofreading 3'->5' exonuclease activity. Thought to be the main polymerase involved in lagging strand synthesis, though there is still debate about its role[6]. Pol : Also highly processive and has proofreading 3'->5' exonuclease activity. Highly related to pol , and thought to be the main polymerase involved in leading strand synthesis[7], though there is again still debate about its role[6]. , , , and Rev1 are Y-family DNA polymerases and Pol is a B-family DNA polymerase. These polymerases are involved in the bypass of DNA damage.[8] There are also other eukaryotic polymerases known, which are not as well characterized: , , , , and . There are also others, but the nomenclature has become quite jumbled.

None of the eukaryotic polymerases can remove primers (5'->3' exonuclease activity); that function is carried out by other enzymes. Only the polymerases that deal with the elongation ( , and ) have proofreading ability (3'->5' exonuclease). Nuclease is a great advocate for DNA replication

DNA repli ation within the cell

Origins of replication
For a cell to divide, it must first replicate its DNA.[7] 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 (eg. dnaA in E coli' and the Origin Recognition Complex in yeast).[8] 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 to the few=flexibilty/many=durability relationship found in hydrogen bonding. [9] 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 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: ). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these.

Prokaryotic DNA replication

DNA replication in prokaryotes is exemplified in E. coli. It is bi-directional and originates at a single origin of replication (OriC).

Initiation
The initiation of replication is mediated by a protein that binds to a region of the origin known as the DnaA box. In E. coli, there are 5 DnaA boxes, each of which contains a highly conserved 9 bp consensus sequence 5' - TTATCCACA - 3'. Binding of DnaA to this region causes it to become negatively supercoiled. Following this, a region of OriC upstream of the DnaA boxes (known as DnaB boxes) become melted. There are three of these regions, and each are 13 bp long, and AT-rich (which facilitates melting because less energy is required to break the two hydrogen bonds that form between A and T nucleotides). This region has the consensus sequence 5' - GATCTNTTNTTTT - 3. Melting of the DnaB boxes requires ATP (which is hydrolyzed by DnaA). Following melting, DnaA recruits a hexameric helicase (six DnaB proteins) to opposite ends of the melted DNA. This is where the replication fork will form. Recruitment of helicase requires six DnaC proteins, each of which is attached to one subunit of helicase. Once this complex is formed, an additional five DnaA proteins bind to the original five DnaA proteins to form five DnaA dimers. DnaC is then released, and the prepriming complex is complete. In order for DNA replication to continue, SSB protein is needed to prevent the single strands of DNA from forming any secondary structures and to prevent them from reannealing, and DNA gyrase is needed to relieves the stress (by creating negative supercoils) created by the action of DnaB helicase. The unwinding of DNA by DnaB helicase allows for primase (DnaG) and RNA polymerase to prime each DNA template so that DNA synthesis can begin.

Elongation
Once priming is complete, DNA polymerase III holoenzyme is loaded into the DNA and replication begins. The catalytic mechanism of DNA polymerase III involves the use of two metal ions in the active site, and a region in the active site that can discriminate between deoxynucleotides and ribonucleotides. The metal ions are general divalent cations that help the 3' OH initiate a nucleophilic attack onto the alpha phosphate of the deoxyribonucleotide and orient and stabilize the negatively charged triphosphate on the deoxyribonucleotide. Nucleophilic attack by the 3' OH on the alpha phosphate releases pyrophosphate, which is then subsequently hydrolyzed (by inorganic phosphatase) into two phosphates. This hydrolysis drives DNA synthesis to completion. Furthermore, DNA polymerase III must be able to distinguish between correctly paired bases and incorrectly paired bases. This is accomplished by distinguishing Watson-Crick base pairs through the use of an active site pocket that is complementary in shape to the structure of correctly paired nucleotides. This pocket has a tyrosine residue that is able to form van der Waals interactions with the correctly paired nucleotide. In addition, dsDNA (double stranded DNA) in the active site has a wider and shallower minor groove that permits the formation of

hydrogen bonds with the third nitrogen of purine bases and the second oxygen of pyrimidine bases. Finally, the active site makes extensive hydrogen bonds with the DNA backbone. These interactions result in the DNA polymerase III closing around a correctly paired base. If a base is inserted and incorrectly paired, these interactions could not occur due to disruptions in hydrogen bonding and van der Waals interactions. DNA is read in the 3' 5' direction, therefore, nucleotides are synthesized (or attached to the template strand) in the 5' 3' direction. However, one of the parent strands of DNA is 3' 5' while the other is 5' 3'. To solve this, replication occurs in opposite directions. Heading towards the replication fork, the leading strand in synthesized in a continuous fashion, only requiring one primer. On the other hand, the lagging strand, heading away from the replication fork, is synthesized in a series of short fragments known as Okazaki fragments, consequently requiring many primers. The RNA primers of Okazaki fragments are subsequently degraded by RNAse H and DNA Polymerase I (exonuclease), and the gap (or nicks) are filled with deoxyribonucleotides and sealed by the enzyme ligase.

Termination
Termination of DNA replication in E. coli is completed through the use of termination sequences and the Tus protein. These sequences allow the two replication forks to pass through in only one direction, but not the other. However, these sequences are not required for termination of replication. Regulation of DNA replication is achieved through several mechanisms. Mechanisms involve the ratio of ATP to ADP, of DnaA to the number of DnaA boxes and the hemimethylation and sequestering of OriC. The ratio of ATP to ADP indicates that the cell has reached a specific size and is ready to divide. This "signal" occurs because in a rich medium, the cell will grow quickly and will have a lot of excess DNA. Furthermore, DnaA binds equally well to ATP or ADP, and only the DnaA-ATP complex is able to initiate replication. Thus, in a fast growing cell, there will be more DnaA-ATP than DnaA-ADP. Because the levels of DnaA are strictly regulated, and 5 DnaA-DnaA dimers are needed to initiate replication, the ratio of DnaA to the number of DnaA boxes in the cell is important. After DNA replication is complete, this number is halved, thus DNA replication cannot occur until the levels of DnaA protein increases. Finally, DNA is sequestered to a membrane-binding protein called SeqA. This protein binds to hemi-methylated GATC DNA sequences. This four bp sequences occurs 11 times in OriC, and newly synthesized DNA only has its parent strand methylated. DAM methyltransferase methylates the newly synthesized strand of DNA only if it is not bound to SeqA. The importance of hemi-methylation is twofold. Firstly, OriC becomes inaccessible to DnaA, and secondly, DnaA binds better to fully methylated DNA than hemimethylated DNA.

Eukaryotic DNA replication

DNA replication in eukaryotes is much more complicated than in prokaryotes, although there are many similar aspects. Eukaryotic cells can only initiate DNA replication at a specific point in the cell cycle, the beginning of S phase.

Mechanism
Location in cell cycle
DNA replication in eukaryotes occurs only in the S phase of the cell cycle. However, preinitiation occurs in the G1 phase. Initiation of replication can only occur during the S-phase. Thus, the separation of pre-initiation and activation ensures that the origin can only fire once per cell cycle. Due to the sheer size of chromosomes in eukaryotes, eukaryotic chromosomes contain multiple origins of replication. Some origins are well characterized, such as the autonomously replicating sequences (ARS) of yeast while other eukaryotic origins, particularly those in metazoa, can be found in spans of thousands of basepairs. However, the assembly and initiation of replicaton is similar in both the protozoa and metazoa.

Preparation in G1 phase
The first step in DNA replication is the formation of the pre-initiation replication complex (the pre-RC). The formation of this complex occurs in two stages. The first stage requires that there is no CDK activity. This can only occur in early G1. The formation of the pre-RC is known as licensing, but a licensed pre-RC cannot initiate replication in the G1 phase Current models hold that it begins with the binding of the origin recognition complex (ORC) to the origin. This complex is a hexamer of related proteins and remains bound to the origin, even after DNA replication occurs. Furthermore, ORC is the functional analogue of prokaryotic DnaA. Following the binding of ORC to the origin, Cdc6/Cdc18 and Cdt1 coordinate the loading of the MCM (Mini Chromosome Maintenance) complex to the origin by first binding to ORC and then binding to the MCM complex. The MCM complex is thought to be the major DNA helicase in eukaryotic organisms. Once binding of MCM occurs, a fully licensed pre-RC exists.

Synthesis in S phase
Activation of the complex occurs in S-phase and requires Cdk2-Cyclin E and Ddk. The activation process begins with the addition of Mcm10 to the pre-RC, which displaces Cdt1. Following this, Ddk phosphorylates Mcm3-7, which activates the helicase. It is believed that ORC and Cdc6/18 are phosphorylated by Cdk2-Cyclin E. Ddk and the Cdk complex then recruits another protein called Cdc45, which then recruits all of the DNA replication proteins to the replication fork. At this stage the origin fires and DNA synthesis begins. Activation of a new round of replication is prevented through the actions of the cyclin dependent kinases and a protein known as geminin. Geminin binds to Cdt1 and sequesters it. It is a periodic protein that first appears in S-phase and is degraded in late M-phase, possibly through the action of the anaphase promoting complex (APC). In addition, phosphorylation of Cdc6/18 prevent it from binding to the ORC (thus inhibiting loading of the MCM complex) while the phosphorylation of ORC remains unclear. Cells in the G0 stage of the cell cycle are prevented from initiating a round of replication because the Mcm proteins are not expressed.

At least four different types of eukaryotic DNA polymerases are involved in the replication of DNA in animal cells (POL , Pol and POL ).
y

Pol forms a complex with a small catalytic (PriS) and a large noncatalytic (PriL) subunit[1], with the Pri subunits acting as a primase (synthesizing an RNA primer), and then with DNA Pol elongating that primer with DNA nucleotides. After around 20 nucleotides[2] elongation is taken over by Pol (on the leading strand) and (on the lagging strand). Pol : Highly processive and has proofreading 3'->5' exonuclease activity. Thought to be the main polymerase involved in lagging strand synthesis, though there is still debate about its role[3]. Pol : Also highly processive and has proofreading 3'->5' exonuclease activity. Highly related to pol , and thought to be the main polymerase involved in leading strand synthesis[4], though there is again still debate about its role[5].

T e replication fork

Many enzymes are involved in the DNA replication fork. Main article: Replication fork When replicating, the original DNA splits in two, forming two "prongs" which resemble a fork (hence the name "replication fork"). DNA has a ladder-like structure; imagine a ladder broken in half vertically, along the steps. Each half of the ladder now requ a new half to ires match it. Because DNA polymerase can only synthesize a new DNA strand in a 5' to 3' manner, the process of replication goes differently for the two strands comprising the DNA double helix. Leading strand The leading strand is that strand of the DNA double helix that is orientated in a 5' to 3' manner. 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 [10][11] in eukaryotes. Lagging strand The lagging strand is that strand of the DNA double helix that is orientated in a 3' to 5' manner. Because of its orientation, opposite to the working orientation of DNA polymerase

III which is in a 5' to 3' manner, replication of the lagging strand is more complicated than 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 .[12] DNA polymerase III or Pol lengthens the primed segments, forming Okazaki fragments. Primer removal in eukaryotes is also performed by Pol .[13] In prokaryotes, DNA polymerase I "reads" the fragments, removes the RNA using its flap endonuclease domain, 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. 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 rota This te. process results in a build-up of twists in the DNA ahead.[14] 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.[15] 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.

Replication fork

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Scheme of the replication fork. a: template, b: leading strand, c: lagging strand, d: replication fork, e: primer, f: Okazaki fragments 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, that are called the leading and lagging strands. DNA polymerase creates new partners for the two strands by adding nucleotides.

Replication

DNA replication When replicating, the original DNA splits in two, forming two "prongs" which resemble a fork (hence the name "replication fork"). DNA has a ladder-like structure; imagine a ladder broken in half vertically, along the steps. Each half of the ladder now requ a new half to ires match it. Because DNA polymerase can only synthesize a new DNA strand in a 5' to 3' manner, the process of replication goes differently for the two strands comprising the DNA double helix.

Leadin strand

The leadin strand is that template strand of the DNA double helix that is orientated in a 3' to 5' manner. Thus, the new, antiparallel strand being produced will be oriented in the 5' to 3' manner. On the leading strand, a polymerase "reads" the template DNA and addsnucleotides to the 3' end of the newly made DNA strand continuously. This polymerase isDNA polymerase III (DNA Pol III) in prokaryotes and presumably Pol [1][2] in eukaryotes.

La

in strand

The la in strand is that template strand of the DNA double helix that is orientated in a 5' to 3' manner. The newly made strand will be antiparallel, oriented in the 3' to 5' direction. Because of its orientation, the newly made strand is extending opposite to the working orientation of DNA polymerase III which is in a 5' to 3' manner. As a result, replication of the lagging strand is more complicated than 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 .[3] DNA polymerase III or Pol lengthens the primed segments, forming Okazaki fragments. Primer removal in eukaryotes is also performed by Pol .[4] In prokaryotes, DNA polymerase I "reads" the fragments, removes the RNA using its flap endonuclease domain, 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.

Re ulation 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 throug h complex interactions between various proteins, includingcyclins and cyclin-dependent kinases.[16]

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.[17] 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.[18] 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.

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.[19] 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, the enzyme 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.

Rollin circle replication

Rolling circle replication Main article: Rolling circle replication Another method of copying DNA, sometimes used in vivo by bacteria and viruses, is the process of rolling circle replication.[20] In this form of replication, a single replication fork progresses around a circular molecule to form multiple linear copies of the DNA sequence. In cells, this process can be used to rapidly synthesize multiple copies of plasmids or viral genomes. In the cell, rolling circle replication is initiated by an initiator protein encoded by the plasmid or virus DNA. This protein is able to nick one strand of the double -stranded, circular DNA molecule at a site called the double-strand origin (DSO) and remains bound to the 5' phosphate end of the nicked strand. The free 3' hydroxyl end is released and can serve as a primer for DNA synthesis. Using the unnicked strand as a template, replication proce eds around the circular DNA molecule, displacing the nicked strand as single -stranded DNA. Continued DNA synthesis produces multiple single -stranded linear copies of the original DNA in a continuous head-to-tail series. In vivo these linear copies are subsequently converted to double-stranded circular molecules. Rolling circle replication can also be performed in vitro and has found wide uses in academic research and biotechnology, often used for amplification of DNA from very small amounts of starting material. Replication can be initiated by nicking a double -stranded circular DNA molecule or by hybridizing a primer to a single-stranded circle of DNA. The use of a reverse primer (or random primers) produces hyperbranched rolling circle amplification, resulting in exponential rather than linear growth of the DNA molecule.

Polymerase chain reaction

Main article: Polymera e c ain 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 thermostableDNA 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]