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DNA Replication - Biology

DNA Replication - Biology


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1. Description of DNA Replication

The human body produces billions of new cells every day. But before undergoing cell division, a cell must first copy the genetic information contained in the cell nucleus – its DNA. In just 6-8 hours a cell is able to copy its entire genome. To accomplish this, DNA replication begins at multiple locations along each chromosome. As the two DNA strands are pulled apart, copying begins at the rate of about 50 nucleotides per second!

DNA Basics

DNA encodes the information needed to build proteins, to regulate physiological processes and maintain homeostasis in our bodies. The basic chemical components of DNA are phosphate, deoxyribose (a sugar) and 4 nitrogenous bases: adenine (A), guanine (G), cytosine (C) and thymine (T).

DNA is a double stranded molecule with the two strands lying antiparallel to each other (they lie parallel to each other but run in opposite directions). The two strands of DNA wind together to form a double helix – a structure that looks similar to a spiral staircase.

Figure (PageIndex{1}). the chemical structure of DNA (CC BY-NC-SA; Madprime)

Figure (PageIndex{2}). semi-conservative replication (CC BY-NC-SA; Madprime)

The strands of DNA are also complementary to each other due to specific base pairing between the two strands: adenine on one strand will always pair with thymine on the opposite strand and cytosine will always bind with guanine.

Figure (PageIndex{3}). DNA replication (CC BY-NC-SA; LadyofHats)

During the process of DNA replication, the parental DNA unwinds and since nucleotides have exclusive partners, each DNA can act as its own template for replication. The two new DNA molecules each consist of one parental strand and one newly made strand. This is known as semiconservative DNA replication.

Figure (PageIndex{4}). DNA double helix (CC BY-NC-SA; Forluvoft)

Important Players in DNA Replication

DNA Helicase: breaks the hydrogen bonds between DNA strands (unwinds DNA)

Topoisomerase: alleviates positive supercoiling (twisting of DNA) ahead of the replication fork

Single-stranded binding proteins (SSBPs): keeps the parental strands apart

Primase: synthesizes an RNA primer (gets synthesis of new strand started)

DNA Polymerase III: synthesizes a daughter strand of DNA

DNA Polymerase I: excises the RNA primers and fills in with DNA

DNA ligase: covalently links the Okazaki fragments together

Mechanism of DNA Replication

Getting replication started

The replication of DNA begins at special sites called origins of replication. Bacteria, which have relatively small circular chromosomes, contain just a single origin of replication. Eukaryotic chromosomes, which are long, linear strands of DNA contain many origins of replication along the DNA strands (see below). Proteins that recognize the origins of replication bind to these sequences and separate the two strands, opening up a replication “bubble”. Replication then proceeds in both directions until the entire molecule is copied.

Figure (PageIndex{5}). replication bubbles (CC BY-NC-SA; Boumphreyfr)

At the end of each replication bubble is a replication fork, a Y shaped region where the parental strands of DNA are unwound so that the replication machinery can copy the DNA. Helicases are enzymes that are responsible for untwisting the double helix at the replication forks, separating the two strands and making them available to serve as templates for DNA replication. The untwisting of the double helix by helicase causes additional twisting of the DNA molecule ahead of the replication fork. Topoisomerase is the enzyme that helps to relieve this strain by breaking, untwisting and rejoining the DNA strands. After the parental strands have been separated by helicase, single stranded binding proteins bind to the parental strands to keep them from reannealing to each other.

Figure (PageIndex{6}). replication fork forming (CC BY-NC-SA; César Benito Jiménez)

The unwound parental strands are now ready to be copied, but DNA Polymerase, the enzyme responsible for copying DNA, cannot initiate the synthesis of DNA, it can only add nucleotides onto an existing chain of nucleotides. To solve this problem, RNA primase puts down a short sequence of RNA complementary to the template DNA, called a primer that can be used to get DNA replication started. DNA polymerase is then able to synthesize new DNA by adding nucleotides to this preexisting chain.

Making a new DNA strand

As mentioned above, DNA polymerases catalyze the synthesis of new DNA by adding nucleotides to the RNA primer that was laid down by RNA Primase. It should be mentioned that in E.coli there are several different DNA polymerases, but two appear to play major roles: DNA Pol III and DNA Pol I. In eukaryotes, the situation is more complex, with at least 11 different DNA polymerases discovered so far, but the general principles we will discuss in this tutorial are applicable to both systems.

In E.coli, DNA Pol III adds a DNA nucleotide to the 3' end of the RNA primer and then continues adding DNA nucleotides complementary to the template strand. As was mentioned previously, the two ends of a DNA strand are different, in that they are antiparallel to each other. This directionality is important for the synthesis of DNA, because DNA polymerase can only add nucleotides to the 3’ end of a growing DNA strand (not the 5’ end). Thus, a new DNA strand can only be made in the 5’ to 3’ direction.

Leading and Lagging Strands

Figure (PageIndex{7}). replication fork (CC BY-NC-SA; César Benito Jiménez)

If we look at the replication fork, we see that this 5’ to 3’ directionality poses a problem. Along one of the template strands, DNA Pol III can synthesize a complementary strand continuously by elongating the new DNA in the 5’ to 3’ direction. This is called the leading strand and it requires only one RNA primer to be made to start replication. However, to elongate the other strand in the 5’ to 3’ direction, DNA Pol III must elongate the new DNA strand in a direction opposite to the movement of the replication fork. This strand is known as the lagging strand. As the replication form proceeds and exposes a new section of the lagging strand template, RNA Primase must put down a new primer for DNA Pol III to elongate. In this way, the lagging strand is completed in a discontinuous manner. The synthesized DNA fragments on the lagging strand are called Okazaki fragments. Another DNA polymerase, DNA Pol I is responsible for removing the RNA primers and replacing them with DNA. DNA ligase then seals the gaps between the Okazaki fragments to complete the newly synthesized lagging strand.

Figure (PageIndex{8}). (CC BY-NC-SA)


DNA Replication Tutorial by Dr. Katherine Harris is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

This tutorial was funded by the Title V-STEM Grant #P031S090007.


DNA Replication

1. DNA helicase (enzyme) unwinds the DNA. The junction is called a replication fork.
2. DNA polymerase adds the complementary nucleotides and binds the sugars and phosphates. DNA polymerase travels from the 3' to the 5' end. The DNA is called the template strand.
3. DNA polymerase adds complementary nucleotides on the other side of the ladder. Traveling in the opposite direction.
4. One side is the leading strand - it follows the helicase as it unwinds.
5. The other side is the lagging strand - its moving away from the helicase (in the 5' to 3' direction).

Replication is called semi-conservative, because one half of the original strand is always saved, or "conserved"

Problem: it reaches the replication fork, but the helicase is moving in the opposite direction. It stops, and another polymerase binds farther down the chain.

This process creates several fragments, called Okazaki Fragments, that are bound together by DNA ligase.

6. During replication, there are many points along the DNA that are synthesized at the same time (multiple replication forks). It would take forever to go from one end to the other, it is more efficient to open up several points at one time.

Replication Errors – can cause a genetic MUTATION
-- PROOFREADING by the polymerase prevents mismatches
-- DNA REPAIR ENZYMES can repair damaged DNA also

/>This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.


Contents

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

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

The pairing of complementary bases in DNA (through hydrogen bonding) means that the information contained within each strand is redundant. Phosphodiester (intra-strand) bonds are stronger than hydrogen (inter-strand) bonds. The actual job of the Phosphodiester bonds is where in DNA polymers connect the 5' carbon of one nucleotide to the 3' carbon of another nucleotide, while the hydrogen bonds stabilize DNA double helices across the helix axis but not in the direction of the axis 1. [11] 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. [12]

DNA polymerases are a family of enzymes that carry out all forms of DNA replication. [14] DNA polymerases in general cannot initiate synthesis of new strands, but can only extend an existing DNA or RNA strand paired with a template strand. To begin synthesis, a short fragment of RNA, called a primer, must be created and paired with the template DNA strand.

DNA polymerase adds a new strand of DNA by extending the 3′ end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from hydrolysis of the high-energy phosphate (phosphoanhydride) bonds between the three phosphates attached to each unincorporated base. Free bases with their attached phosphate groups are called nucleotides in particular, bases with three attached phosphate groups are called nucleoside triphosphates. When a nucleotide is being added to a growing DNA strand, the formation of a phosphodiester bond between the proximal phosphate of the nucleotide to the growing chain is accompanied by hydrolysis of a high-energy phosphate bond with release of the two distal 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. [Note 1]

In general, DNA polymerases are highly accurate, with an intrinsic error rate of less than one mistake for every 10 7 nucleotides added. [15] In addition, some 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 for errors, being capable of distinguishing mismatches in the newly synthesized DNA strand from the original strand sequence. Together, these three discrimination steps enable replication fidelity of less than one mistake for every 10 9 nucleotides added. [15]

The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected E. coli. [16] During the period of exponential DNA increase at 37 °C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 10 8 . [17]

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

Initiation Edit

For a cell to divide, it must first replicate its DNA. [18] DNA replication is an all-or-none process once replication begins, it proceeds to completion. Once replication is complete, it does not occur again in the same cell cycle. This is made possible by the division of initiation of the pre-replication complex.

Pre-replication complex Edit

In late mitosis and early G1 phase, a large complex of initiator proteins assembles into the pre-replication complex at particular points in the DNA, known as "origins". [7] In E. coli the primary initiator protein is DnaA in yeast, this is the origin recognition complex. [19] 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. [20] In eukaryotes, the origin recognition complex catalyzes the assembly of initiator proteins into the pre-replication complex. Cdc6 and Cdt1 then associate with the bound origin recognition complex at the origin in order to form a larger complex necessary to load the Mcm complex onto the DNA. The Mcm complex is the helicase that will unravel the DNA helix at the replication origins and replication forks in eukaryotes. The Mcm complex is recruited at late G1 phase and loaded by the ORC-Cdc6-Cdt1 complex onto the DNA via ATP-dependent protein remodeling. The loading of the Mcm complex onto the origin DNA marks the completion of pre-replication complex formation. [21]

If environmental conditions are right in late G1 phase, the G1 and G1/S cyclin-Cdk complexes are activated, which stimulate expression of genes that encode components of the DNA synthetic machinery. G1/S-Cdk activation also promotes the expression and activation of S-Cdk complexes, which may play a role in activating replication origins depending on species and cell type. Control of these Cdks vary depending cell type and stage of development. This regulation is best understood in budding yeast, where the S cyclins Clb5 and Clb6 are primarily responsible for DNA replication. [22] Clb5,6-Cdk1 complexes directly trigger the activation of replication origins and are therefore required throughout S phase to directly activate each origin. [21]

In a similar manner, Cdc7 is also required through S phase to activate replication origins. Cdc7 is not active throughout the cell cycle, and its activation is strictly timed to avoid premature initiation of DNA replication. In late G1, Cdc7 activity rises abruptly as a result of association with the regulatory subunit Dbf4, which binds Cdc7 directly and promotes its protein kinase activity. Cdc7 has been found to be a rate-limiting regulator of origin activity. Together, the G1/S-Cdks and/or S-Cdks and Cdc7 collaborate to directly activate the replication origins, leading to initiation of DNA synthesis. [21]

Preinitiation complex Edit

In early S phase, S-Cdk and Cdc7 activation lead to the assembly of the preinitiation complex, a massive protein complex formed at the origin. Formation of the preinitiation complex displaces Cdc6 and Cdt1 from the origin replication complex, inactivating and disassembling the pre-replication complex. Loading the preinitiation complex onto the origin activates the Mcm helicase, causing unwinding of the DNA helix. The preinitiation complex also loads α-primase and other DNA polymerases onto the DNA. [21]

After α-primase synthesizes the first primers, the primer-template junctions interact with the clamp loader, which loads the sliding clamp onto the DNA to begin DNA synthesis. The components of the preinitiation complex remain associated with replication forks as they move out from the origin. [21]

Elongation Edit

DNA polymerase has 5′–3′ activity. All known DNA replication systems require a free 3′ hydroxyl group before synthesis can be initiated (note: the DNA template is read in 3′ to 5′ direction whereas a new strand is synthesized in the 5′ to 3′ direction—this is often confused). Four distinct mechanisms for DNA synthesis are recognized:

  1. All cellular life forms and many DNA viruses, phages and plasmids use a primase to synthesize a short RNA primer with a free 3′ OH group which is subsequently elongated by a DNA polymerase.
  2. The retroelements (including retroviruses) employ a transfer RNA that primes DNA replication by providing a free 3′ OH that is used for elongation by the reverse transcriptase.
  3. In the adenoviruses and the φ29 family of bacteriophages, the 3′ OH group is provided by the side chain of an amino acid of the genome attached protein (the terminal protein) to which nucleotides are added by the DNA polymerase to form a new strand.
  4. In the single stranded DNA viruses—a group that includes the circoviruses, the geminiviruses, the parvoviruses and others—and also the many phages and plasmids that use the rolling circle replication (RCR) mechanism, the RCR endonuclease creates a nick in the genome strand (single stranded viruses) or one of the DNA strands (plasmids). The 5′ end of the nicked strand is transferred to a tyrosine residue on the nuclease and the free 3′ OH group is then used by the DNA polymerase to synthesize the new strand.

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 primers to the template strands. The leading strand receives one RNA primer while the lagging strand receives several. The leading strand is continuously extended from the primer by a DNA polymerase with high processivity, while the lagging strand is extended discontinuously from each primer forming Okazaki fragments. RNase removes the primer RNA fragments, and a low processivity DNA polymerase distinct from the replicative polymerase enters to fill the gaps. When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. Ligase works to fill these nicks in, thus completing the newly replicated DNA molecule.

The primase used in this process differs significantly between bacteria and archaea/eukaryotes. Bacteria use a primase belonging to the DnaG protein superfamily which contains a catalytic domain of the TOPRIM fold type. [23] The TOPRIM fold contains an α/β core with four conserved strands in a Rossmann-like topology. This structure is also found in the catalytic domains of topoisomerase Ia, topoisomerase II, the OLD-family nucleases and DNA repair proteins related to the RecR protein.

The primase used by archaea and eukaryotes, in contrast, contains a highly derived version of the RNA recognition motif (RRM). This primase is structurally similar to many viral RNA-dependent RNA polymerases, reverse transcriptases, cyclic nucleotide generating cyclases and DNA polymerases of the A/B/Y families that are involved in DNA replication and repair. In eukaryotic replication, the primase forms a complex with Pol α. [24]

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

In eukaryotes, the low-processivity enzyme, Pol α, helps to initiate replication because it forms a complex with primase. [25] 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. [26] Primer removal is completed Pol δ [27] while repair of DNA during replication is completed by Pol ε.

As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming a replication fork with two prongs. In bacteria, which have a single origin of replication on their circular chromosome, this process creates a "theta structure" (resembling the Greek letter theta: θ). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these. [28]

Replication fork Edit

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

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

Leading strand Edit

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

Lagging strand Edit

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

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

Dynamics at the replication fork Edit

In all cases the helicase is composed of six polypeptides that wrap around only one strand of the DNA being replicated. The two polymerases are bound to the helicase heximer. In eukaryotes the helicase wraps around the leading strand, and in prokaryotes it wraps around the lagging strand. [29]

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. [30] 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 helix topoisomerases (including DNA gyrase) achieve this by adding negative supercoils to the DNA helix. [31]

Bare single-stranded DNA tends to fold back on itself forming secondary structures these structures can interfere with the movement of DNA polymerase. To prevent this, single-strand binding proteins bind to the DNA until a second strand is synthesized, preventing secondary structure formation. [32]

Double-stranded DNA is coiled around histones that play an important role in regulating gene expression so the replicated DNA must be coiled around histones at the same places as the original DNA. To ensure this, histone chaperones disassemble the chromatin before it is replicated and replace the histones in the correct place. Some steps in this reassembly are somewhat speculative. [33]

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 to be threaded through it. Once the polymerase reaches the end of the template or detects double-stranded DNA, the sliding clamp undergoes a conformational change that releases the DNA polymerase. Clamp-loading proteins are used to initially load the clamp, recognizing the junction between template and RNA primers. [6] :274-5

DNA replication proteins Edit

At the replication fork, many replication enzymes assemble on the DNA into a complex molecular machine called the replisome. The following is a list of major DNA replication enzymes that participate in the replisome: [34]

Enzyme Function in DNA replication
DNA helicase Also known as helix destabilizing enzyme. Helicase separates the two strands of DNA at the Replication Fork behind the topoisomerase.
DNA polymerase The enzyme responsible for catalyzing the addition of nucleotide substrates to DNA in the 5′ to 3′ direction during DNA replication. Also performs proof-reading and error correction. There exist many different types of DNA Polymerase, each of which perform different functions in different types of cells.
DNA clamp A protein which prevents elongating DNA polymerases from dissociating from the DNA parent strand.
Single-strand DNA-binding protein Bind to ssDNA and prevent the DNA double helix from re-annealing after DNA helicase unwinds it, thus maintaining the strand separation, and facilitating the synthesis of the new strand.
Topoisomerase Relaxes the DNA from its super-coiled nature.
DNA gyrase Relieves strain of unwinding by DNA helicase this is a specific type of topoisomerase
DNA ligase Re-anneals the semi-conservative strands and joins Okazaki Fragments of the lagging strand.
Primase Provides a starting point of RNA (or DNA) for DNA polymerase to begin synthesis of the new DNA strand.
Telomerase Lengthens telomeric 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. [35]

Replication machinery Edit

Replication machineries consist of factors involved in DNA replication and appearing on template ssDNAs. Replication machineries include primosotors are replication enzymes DNA polymerase, DNA helicases, DNA clamps and DNA topoisomerases, and replication proteins e.g. single-stranded DNA binding proteins (SSB). In the replication machineries these components coordinate. In most of the bacteria, all of the factors involved in DNA replication are located on replication forks and the complexes stay on the forks during DNA replication. These replication machineries are called replisomes or 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. [36] In an alternative figure, DNA factories are similar to projectors and DNAs are like as cinematic films passing constantly into the projectors. In the replication factory model, after both DNA helicases for leading strands and lagging strands are loaded on the template DNAs, the helicases run along the DNAs into each other. The helicases remain associated for the remainder of replication process. Peter Meister et al. observed directly replication sites in budding yeast by monitoring green fluorescent protein (GFP)-tagged DNA polymerases α. They detected DNA replication of pairs of the tagged loci spaced apart symmetrically from a replication origin and found that the distance between the pairs decreased markedly by time. [37] This finding suggests that the mechanism of DNA replication goes with DNA factories. That is, couples of replication factories are loaded on replication origins and the factories associated with each other. Also, template DNAs move into the factories, which bring extrusion of the template ssDNAs and new DNAs. Meister's finding is the first direct evidence of replication factory model. Subsequent research has shown that DNA helicases form dimers in many eukaryotic cells and bacterial replication machineries stay in single intranuclear location during DNA synthesis. [36]

The replication factories perform disentanglement of sister chromatids. The disentanglement is essential for distributing the chromatids into daughter cells after DNA replication. Because sister chromatids after DNA replication hold each other by Cohesin rings, there is the only chance for the disentanglement in DNA replication. Fixing of replication machineries as replication factories can improve the success rate of DNA replication. If replication forks move freely in chromosomes, catenation of nuclei is aggravated and impedes mitotic segregation. [37]

Termination Edit

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

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

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. E. coli regulates this process through the use of termination sequences that, when bound by the Tus protein, enable only one direction of replication fork to pass through. As a result, the replication forks are constrained to always meet within the termination region of the chromosome. [38]

Eukaryotes Edit

Within eukaryotes, DNA replication is controlled within the context of the cell cycle. As the cell grows and divides, it progresses through stages in the cell cycle DNA replication takes place during the S phase (synthesis phase). The progress of the eukaryotic cell through the cycle is controlled by cell cycle checkpoints. Progression through checkpoints is controlled through complex interactions between various proteins, including cyclins and cyclin-dependent kinases. [39] Unlike bacteria, eukaryotic DNA replicates in the confines of the nucleus. [40]

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

After passing through the G1/S checkpoint, DNA must be replicated only once in each cell cycle. When the Mcm complex moves away from the origin, the pre-replication complex is dismantled. Because a new Mcm complex cannot be loaded at an origin until the pre-replication subunits are reactivated, one origin of replication can not be used twice in the same cell cycle. [21]

Activation of S-Cdks in early S phase promotes the destruction or inhibition of individual pre-replication complex components, preventing immediate reassembly. S and M-Cdks continue to block pre-replication complex assembly even after S phase is complete, ensuring that assembly cannot occur again until all Cdk activity is reduced in late mitosis. [21]

In budding yeast, inhibition of assembly is caused by Cdk-dependent phosphorylation of pre-replication complex components. At the onset of S phase, phosphorylation of Cdc6 by Cdk1 causes the binding of Cdc6 to the SCF ubiquitin protein ligase, which causes proteolytic destruction of Cdc6. Cdk-dependent phosphorylation of Mcm proteins promotes their export out of the nucleus along with Cdt1 during S phase, preventing the loading of new Mcm complexes at origins during a single cell cycle. Cdk phosphorylation of the origin replication complex also inhibits pre-replication complex assembly. The individual presence of any of these three mechanisms is sufficient to inhibit pre-replication complex assembly. However, mutations of all three proteins in the same cell does trigger reinitiation at many origins of replication within one cell cycle. [21] [41]

In animal cells, the protein geminin is a key inhibitor of pre-replication complex assembly. Geminin binds Cdt1, preventing its binding to the origin recognition complex. In G1, levels of geminin are kept low by the APC, which ubiquitinates geminin to target it for degradation. When geminin is destroyed, Cdt1 is released, allowing it to function in pre-replication complex assembly. At the end of G1, the APC is inactivated, allowing geminin to accumulate and bind Cdt1. [21]

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

Replication focus Edit

In vertebrate cells, replication sites concentrate into positions called replication foci. [37] 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., [37] (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. [37] Traditionally, replication sites were fixed on spatial structure of chromosomes by nuclear matrix or lamins. The Heun's results denied the traditional concepts, budding yeasts do not have lamins, and support that replication origins self-assemble and form replication foci.

By firing of replication origins, controlled spatially and temporally, the formation of replication foci is regulated. D. A. Jackson et al.(1998) revealed that neighboring origins fire simultaneously in mammalian cells. [37] 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 do not fire in normal DNA replication.

Bacteria Edit

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. [42] In E. coli, the best-characterized bacteria, DNA replication is regulated through several mechanisms, including: the hemimethylation and sequestering of the origin sequence, the ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP), and the levels of protein DnaA. All these control the binding of initiator proteins to the origin sequences.

Because E. coli methylates GATC DNA sequences, DNA synthesis results in hemimethylated sequences. This hemimethylated DNA is recognized by the protein SeqA, which binds and sequesters the origin sequence in addition, DnaA (required for initiation of replication) binds less well to hemimethylated DNA. As a result, newly replicated origins are prevented from immediately initiating another round of DNA replication. [43]

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

In fast-growing bacteria, such as E. coli, chromosome replication takes more time than dividing the cell. The bacteria solve this by initiating a new round of replication before the previous one has been terminated. [44] The new round of replication will form the chromosome of the cell that is born two generations after the dividing cell. This mechanism creates overlapping replication cycles.


DNA Replication in Prokaryotes

Recall that the prokaryotic chromosome is a circular molecule with a less extensive coiling structure than eukaryotic chromosomes. The eukaryotic chromosome is linear and highly coiled around proteins. While there are many similarities in the DNA replication process, these structural differences necessitate some differences in the DNA replication process in these two life forms.

DNA replication has been extremely well-studied in prokaryotes, primarily because of the small size of the genome and large number of variants available. Escherichia coli has 4.6 million base pairs in a single circular chromosome, and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the chromosome in both directions. This means that approximately 1000 nucleotides are added per second. The process is much more rapid than in eukaryotes. [link] summarizes the differences between prokaryotic and eukaryotic replications.

Differences between Prokaryotic and Eukaryotic Replications
Property Prokaryotes Eukaryotes
Origin of replication Single Multiple
Rate of replication 1000 nucleotides/s 50 to 100 nucleotides/s
Chromosome structure circular linear
Telomerase Not present Present

Click through a tutorial on DNA replication.


Bacteria

The single molecule of DNA that is the E. coli genome contains 4.7 x 10 6 nucleotide pairs. DNA replication begins at a single, fixed location in this molecule, the replication origin, proceeds at about 1000 nucleotides per second, and thus is done in no more than 40 minutes. And thanks to the precision of the process (which includes a "proof-reading" function), the job is done with only about one incorrect nucleotide for every 10 9 nucleotides inserted. In other words, more often than not, the E. coli genome (4.7 x 10 6 ) is copied without error!

Eukaryotes

The average human chromosome contains 150 x 10 6 nucleotide pairs which are copied at about 50 base pairs per second. The process would take a month (rather than the hour it actually does) but for the fact that there are thousands of places on the eukaryotic chromosome, called origins of replication, where replication can begin and then proceed in both directions. Replication begins at some replication origins earlier in S phase than at others, but the process is completed for all by the end of S phase. As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, finally forming two new molecules.


The end replication problem

The limitations of the polymerase enzyme come to a head at the end of DNA replication, when, travelling along the template 5’-3’ strand, the enzyme at last reaches the strand’s 3’ riboxyl end. The DNA polymerase enzyme requires RNA fragments to begin replication, and there is nothing for such a fragment to attach to at the 3’ end of the DNA strand. Therefore, with every round of replication, a small fragment of the DNA is lost from the end of the chromosome, since it cannot be replicated. The cell solves this problem by having telomeres at the ends of chromosomes, where they prevent the loss of valuable genetic information by acting as a disposable buffer. Over time, with each successive round of DNA replication, the telomeric DNA shortens until finally there is no more disposable buffer, at which point the cell stops dividing and enters senescence. For a visualisation of the end replication problem check out this YouTube video:


DNA polymerase can make mistakes while adding nucleotides. It edits the DNA by proofreading every newly added base. Incorrect bases are removed and replaced by the correct base, and then polymerization continues (Figure 3a). Most mistakes are corrected during replication, although when this does not happen, the mismatch repair mechanism is employed. Mismatch repair enzymes recognize the wrongly incorporated base and excise it from the DNA, replacing it with the correct base (Figure 3b). In yet another type of repair, nucleotide excision repair, the DNA double strand is unwound and separated, the incorrect bases are removed along with a few bases on the 5′ and 3′ end, and these are replaced by copying the template with the help of DNA polymerase (Figure 3c). Nucleotide excision repair is particularly important in correcting thymine dimers, which are primarily caused by ultraviolet light. In a thymine dimer, two thymine nucleotides adjacent to each other on one strand are covalently bonded to each other rather than their complementary bases. If the dimer is not removed and repaired it will lead to a mutation. Individuals with flaws in their nucleotide excision repair genes show extreme sensitivity to sunlight and develop skin cancers early in life.

Figure 3. Proofreading by DNA polymerase (a) corrects errors during replication. In mismatch repair (b), the incorrectly added base is detected after replication. The mismatch repair proteins detect this base and remove it from the newly synthesized strand by nuclease action. The gap is now filled with the correctly paired base. Nucleotide excision (c) repairs thymine dimers. When exposed to UV, thymines lying adjacent to each other can form thymine dimers. In normal cells, they are excised and replaced.

Most mistakes are corrected if they are not, they may result in a mutation—defined as a permanent change in the DNA sequence. Mutations in repair genes may lead to serious consequences like cancer.


14.1 | Historical Basis of Modern Understanding

By the end of this section, you will be able to:

  • Explain transformation of DNA.
  • Describe the key experiments that helped identify that DNA is the genetic material.
  • State and explain Chargaff’s rules

Modern understandings of DNA have evolved from the discovery of nucleic acids to the development of the double-helix model. In the 1860s, Friedrich Miescher (Figure 14.2), a physician by profession, was the first person to isolate phosphate- rich chemicals from white blood cells or leukocytes. He named these chemicals (which would eventually be known as RNA and DNA) nuclein because they were isolated from the nuclei of the cells.

Figure 14.2 Friedrich Miescher (1844–1895) discovered nucleic acids.

A half century later, British bacteriologist Frederick Griffith was perhaps the first person to show that hereditary information could be transferred from one cell to another “horizontally,” rather than by descent. In 1928, he reported the first demonstration of bacterial transformation, a process in which external DNA is taken up by a cell, thereby changing morphology and physiology. He was working with Streptococcus pneumoniae, the bacterium that causes pneumonia. Griffith worked with two strains, rough (R) and smooth (S). The R strain is non-pathogenic (does not cause disease) and is called rough because its outer surface is a cell wall and lacks a capsule as a result, the cell surface appears uneven under the microscope. The S strain is pathogenic (disease-causing) and has a capsule outside its cell wall. As a result, it has a smooth appearance under the microscope. Griffith injected the live R strain into mice and they survived. In another experiment, when he injected mice with the heat-killed S strain, they also survived. In a third set of experiments, a mixture of live R strain and heat-killed S strain were injected into mice, and—to his surprise—the mice died. Upon isolating the live bacteria from the dead mouse, only the S strain of bacteria was recovered. When this isolated S strain was injected into fresh mice, the mice died. Griffith concluded that something had passed from the heat-killed S strain into the live R strain and transformed it into the pathogenic S strain, and he called this the transforming principle (Figure 11.3). These experiments are now famously known as Griffith’s transformation experiments.

Figure 14.3 Two strains of S.pneumoniae were used in Griffith’s transformation experiments. The R strain is non- pathogenic. The S strain is pathogenic and causes death. When Griffith injected a mouse with the heat-killed S strain and a live R strain, the mouse died. The S strain was recovered from the dead mouse. Thus, Griffith concluded that something had passed from the heat-killed S strain to the R strain, transforming the R strain into S strain in the process. (credit “living mouse”: modification of work by NIH credit “dead mouse”: modification of work by Sarah Marriage)

Scientists Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944) were interested in exploring this transforming principle further. They isolated the S strain from the dead mice and isolated the proteins and nucleic acids, namely RNA and DNA, as these were possible candidates for the molecule of heredity. They conducted a systematic elimination study. They used enzymes that specifically degraded each component and then used each mixture separately to transform the R strain. They found that when DNA was degraded, the resulting mixture was no longer able to transform the bacteria, whereas all of the other combinations were able to transform the bacteria. This led them to conclude that DNA was the transforming principle.

Experiments conducted by Martha Chase and Alfred Hershey in 1952 provided confirmatory evidence that DNA was the genetic material and not proteins. Chase and Hershey were studying a bacteriophage, which is a virus that infects bacteria. Viruses typically have a simple structure: a protein coat, called the capsid, and a nucleic acid core that contains the genetic material, either DNA or RNA. The bacteriophage infects the host bacterial cell by attaching to its surface, and then it injects its nucleic acids inside the cell. The phage DNA makes multiple copies of itself using the host machinery, and eventually the host cell bursts, releasing a large number of bacteriophages. Hershey and Chase labeled one batch of phage with radioactive sulfur, 35 S, to label the protein coat. Another batch of phage were labeled with radioactive phosphorus, 32 P. Because phosphorous is found in DNA, but not protein, the DNA and not the protein would be tagged with radioactive phosphorus.

Each batch of phage was allowed to infect the cells separately. After infection, the phage bacterial suspension was put in a blender, which caused the phage coat to be detached from the host cell. The phage and bacterial suspension was spun down in a centrifuge. The heavier bacterial cells settled down and formed a pellet, whereas the lighter phage particles stayed in the supernatant (the liquid above the pellet). In the tube that contained phage labeled with 35 S, the supernatant contained the radioactively labeled phage, whereas no radioactivity was detected in the pellet. In the tube that contained the phage labeled with 32 P, the radioactivity was detected in the pellet that contained the heavier bacterial cells, and no radioactivity was detected in the supernatant. Hershey and Chase concluded that it was the phage DNA that was injected into the cell and carried information to produce more phage particles, thus providing evidence that DNA was the genetic material and not proteins (Figure 14.4).

Figure 14.4 In Hershey and Chase’s experiments, bacteria were infected with phage radiolabeled with either 35S, which labels protein, or 32P, which labels DNA. Only 32P entered the bacterial cells, indicating that DNA is the genetic material.

Around this same time, Austrian biochemist Erwin Chargaff examined the content of DNA in different species and found that the amounts of adenine, thymine, guanine, and cytosine were not found in equal quantities, and that it varied from species to species, but not between individuals of the same species. He found that the amount of adenine equals the amount of thymine, and the amount of cytosine equals the amount of guanine, or A = T and G = C. These are also known as Chargaff’s rules. This finding proved immensely useful when Watson and Crick were getting ready to propose their DNA double helix model, discussed in Chapter 5.


Telomerase and Aging

Cells that undergo cell division continue to have their telomeres shortened because most somatic cells do not make telomerase. This essentially means that telomere shortening is associated with aging. With the advent of modern medicine, preventative health care, and healthier lifestyles, the human life span has increased, and there is an increasing demand for people to look younger and have a better quality of life as they grow older.

In 2010, scientists found that telomerase can reverse some age-related conditions in mice. This may have potential in regenerative medicine. 1 Telomerase-deficient mice were used in these studies these mice have tissue atrophy, stem cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved the function of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.

Cancer is characterized by uncontrolled cell division of abnormal cells. The cells accumulate mutations, proliferate uncontrollably, and can migrate to different parts of the body through a process called metastasis. Scientists have observed that cancerous cells have considerably shortened telomeres and that telomerase is active in these cells. Interestingly, only after the telomeres were shortened in the cancer cells did the telomerase become active. If the action of telomerase in these cells can be inhibited by drugs during cancer therapy, then the cancerous cells could potentially be stopped from further division.

Difference between Prokaryotic and Eukaryotic Replication
Property Prokaryotes Eukaryotes
Origin of replication Single Multiple
Rate of replication 1000 nucleotides/s 50 to 100 nucleotides/s
DNA polymerase types 5 14
Telomerase Not present Present
RNA primer removal DNA pol I RNase H
Strand elongation DNA pol III Pol δ, pol ε
Sliding clamp Sliding clamp PCNA


Biology 171

By the end of this section, you will be able to do the following:

  • Discuss the similarities and differences between DNA replication in eukaryotes and prokaryotes
  • State the role of telomerase in DNA replication

Eukaryotic genomes are much more complex and larger in size than prokaryotic genomes. Eukaryotes also have a number of different linear chromosomes. The human genome has 3 billion base pairs per haploid set of chromosomes, and 6 billion base pairs are replicated during the S phase of the cell cycle. There are multiple origins of replication on each eukaryotic chromosome humans can have up to 100,000 origins of replication across the genome. The rate of replication is approximately 100 nucleotides per second, much slower than prokaryotic replication. In yeast, which is a eukaryote, special sequences known as autonomously replicating sequences (ARS) are found on the chromosomes. These are equivalent to the origin of replication in E. coli.

The number of DNA polymerases in eukaryotes is much more than in prokaryotes: 14 are known, of which five are known to have major roles during replication and have been well studied. They are known as pol α, pol β, pol γ, pol δ, and pol ε.

The essential steps of replication are the same as in prokaryotes. Before replication can start, the DNA has to be made available as a template. Eukaryotic DNA is bound to basic proteins known as histones to form structures called nucleosomes. Histones must be removed and then replaced during the replication process, which helps to account for the lower replication rate in eukaryotes. The chromatin (the complex between DNA and proteins) may undergo some chemical modifications, so that the DNA may be able to slide off the proteins or be accessible to the enzymes of the DNA replication machinery. At the origin of replication, a pre-replication complex is made with other initiator proteins. Helicase and other proteins are then recruited to start the replication process ((Figure)).

Difference between Prokaryotic and Eukaryotic Replication
Property Prokaryotes Eukaryotes
Origin of replication Single Multiple
Rate of replication 1000 nucleotides/s 50 to 100 nucleotides/s
DNA polymerase types 5 14
Telomerase Not present Present
RNA primer removal DNA pol I RNase H
Strand elongation DNA pol III Pol α, pol δ, pol ε
Sliding clamp Sliding clamp PCNA

A helicase using the energy from ATP hydrolysis opens up the DNA helix. Replication forks are formed at each replication origin as the DNA unwinds. The opening of the double helix causes over-winding, or supercoiling, in the DNA ahead of the replication fork. These are resolved with the action of topoisomerases. Primers are formed by the enzyme primase, and using the primer, DNA pol can start synthesis. Three major DNA polymerases are then involved: α, δ and ε. DNA pol α adds a short (20 to 30 nucleotides) DNA fragment to the RNA primer on both strands, and then hands off to a second polymerase. While the leading strand is continuously synthesized by the enzyme pol δ, the lagging strand is synthesized by pol ε. A sliding clamp protein known as PCNA (proliferating cell nuclear antigen) holds the DNA pol in place so that it does not slide off the DNA. As pol δ runs into the primer RNA on the lagging strand, it displaces it from the DNA template. The displaced primer RNA is then removed by RNase H (AKA flap endonuclease) and replaced with DNA nucleotides. The Okazaki fragments in the lagging strand are joined after the replacement of the RNA primers with DNA. The gaps that remain are sealed by DNA ligase, which forms the phosphodiester bond.

Telomere replication

Unlike prokaryotic chromosomes, eukaryotic chromosomes are linear. As you’ve learned, the enzyme DNA pol can add nucleotides only in the 5′ to 3′ direction. In the leading strand, synthesis continues until the end of the chromosome is reached. On the lagging strand, DNA is synthesized in short stretches, each of which is initiated by a separate primer. When the replication fork reaches the end of the linear chromosome, there is no way to replace the primer on the 5’ end of the lagging strand. The DNA at the ends of the chromosome thus remains unpaired, and over time these ends, called telomeres, may get progressively shorter as cells continue to divide.

Telomeres comprise repetitive sequences that code for no particular gene. In humans, a six-base-pair sequence, TTAGGG, is repeated 100 to 1000 times in the telomere regions. In a way, these telomeres protect the genes from getting deleted as cells continue to divide. The telomeres are added to the ends of chromosomes by a separate enzyme, telomerase ((Figure)), whose discovery helped in the understanding of how these repetitive chromosome ends are maintained. The telomerase enzyme contains a catalytic part and a built-in RNA template. It attaches to the end of the chromosome, and DNA nucleotides complementary to the RNA template are added on the 3′ end of the DNA strand. Once the 3′ end of the lagging strand template is sufficiently elongated, DNA polymerase can add the nucleotides complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.


Telomerase is typically active in germ cells and adult stem cells. It is not active in adult somatic cells. For their discovery of telomerase and its action, Elizabeth Blackburn, Carol W. Greider, and Jack W. Szostak ((Figure)) received the Nobel Prize for Medicine and Physiology in 2009.


Telomerase and Aging

Cells that undergo cell division continue to have their telomeres shortened because most somatic cells do not make telomerase. This essentially means that telomere shortening is associated with aging. With the advent of modern medicine, preventative health care, and healthier lifestyles, the human life span has increased, and there is an increasing demand for people to look younger and have a better quality of life as they grow older.

In 2010, scientists found that telomerase can reverse some age-related conditions in mice. This may have potential in regenerative medicine. 1 Telomerase-deficient mice were used in these studies these mice have tissue atrophy, stem cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved the function of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.

Cancer is characterized by uncontrolled cell division of abnormal cells. The cells accumulate mutations, proliferate uncontrollably, and can migrate to different parts of the body through a process called metastasis. Scientists have observed that cancerous cells have considerably shortened telomeres and that telomerase is active in these cells. Interestingly, only after the telomeres were shortened in the cancer cells did the telomerase become active. If the action of telomerase in these cells can be inhibited by drugs during cancer therapy, then the cancerous cells could potentially be stopped from further division.

Section Summary

Replication in eukaryotes starts at multiple origins of replication. The mechanism is quite similar to that in prokaryotes. A primer is required to initiate synthesis, which is then extended by DNA polymerase as it adds nucleotides one by one to the growing chain. The leading strand is synthesized continuously, whereas the lagging strand is synthesized in short stretches called Okazaki fragments. The RNA primers are replaced with DNA nucleotides the DNA Okazaki fragments are linked into one continuous strand by DNA ligase. The ends of the chromosomes pose a problem as the primer RNA at the 5’ ends of the DNA cannot be replaced with DNA, and the chromosome is progressively shortened. Telomerase, an enzyme with an inbuilt RNA template, extends the ends by copying the RNA template and extending one strand of the chromosome. DNA polymerase can then fill in the complementary DNA strand using the regular replication enzymes. In this way, the ends of the chromosomes are protected.

Free Response

How do the linear chromosomes in eukaryotes ensure that its ends are replicated completely?

Telomerase has an inbuilt RNA template that extends the 3′ end, so primer is synthesized and extended. Thus, the ends are protected.

Footnotes

    Jaskelioff et al., “Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice,” Nature 469 (2011): 102-7.

Glossary


Watch the video: ΑΝΤΙΓΡΑΦΗ DNA 3 - DNA REPLICATION 3 (December 2022).