lymphoid system

Lymphatic system
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Lymphatic System
An image displaying the lymphatic system.

The lymphatic system in vertebrates is a network of conduits that carry a clear fluid called lymph. It also includes the lymphoid tissue through which the lymph travels. Lymphoid tissue is found in many organs, particularly the lymph nodes, and in the lymphoid follicles associated with the digestive system such as the tonsils. The system also includes all the structures dedicated to the circulation and production of lymphocytes, which includes the spleen, thymus, bone marrow and the lymphoid tissue associated with the digestive system.[1] The lymphatic system as we know it today was first described independently by Olaus Rudbeck and Thomas Bartholin.

The blood does not directly come in contact with the parenchymal cells and tissues in the body, but constituents of the blood first exit the microvascular exchange blood vessels to become interstitial fluid, which comes into contact with the parenchymal cells of the body. Lymph is the fluid that is formed when interstitial fluid enters the initial lymphatic vessels of the lymphatic system. The lymph is then moved along the lymphatic vessel network by either intrinsic contractions of the lymphatic vessels or by extrinsic compression of the lymphatic vessels via external tissue forces (e.g. the contractions of skeletal muscles).

The lymphatic system has three interrelated functions. It is responsible for the removal of interstitial fluid from tissues. It absorbs and transports fatty acids and fats as chyle to the circulatory system. The last function of the lymphatic system is the transport of immune cells to and from the lymph nodes. The lymph transports antigen presenting cells (APCs), such as dendritic cells, to the lymph nodes where an immune response is stimulated. The lymph also carries lymphocytes from the efferent lymphatics exiting the lymph nodes.

The study of lymphatic drainage of various organs is important in diagnosis, prognosis, and treatment of cancer. The lymphatic system, because of its physical proximity to many tissues of the body, is responsible for carrying cancerous cells between the various parts of the body in a process called metastasis. The intervening lymph nodes can trap the cancer cells. If they are not successful in destroying the cancer cells the nodes may become sites of secondary tumors.

Diseases and other problems of the lymphatic system can cause swelling and other symptoms. Problems with the system can impair the body's ability to fight infections.
Contents
[hide]

* 1 Organization
* 2 Formation of lymph
* 3 Lymphoid tissue
o 3.1 Lymph nodes
o 3.2 Lymphatics
+ 3.2.1 General structure of Lymphatics
+ 3.2.2 Lymph capillaries
+ 3.2.3 Lymph vessels
* 4 Function of the fatty acid transport system
* 5 Diseases of the lymphatic system
* 6 Development of lymphatic tissue
* 7 History
* 8 See also
* 9 References
* 10 External links

[edit] Organization

The lymphatic system can be broadly divided into the conducting system and the lymphoid tissue.

The conducting system carries the lymph and consists of tubular vessels that include the lymph capillaries, the lymph vessels, and the right and thoracic ducts.

The lymphoid tissue is primarily involved in immune responses and consists of lymphocytes and other white blood cells enmeshed in connective tissue through which the lymph passes. Regions of the lymphoid tissue that are densely packed with lymphocytes are known as lymphoid follicles. Lymphoid tissue can either be structurally well organized as lymph nodes or may consist of loosely organized lymphoid follicles known as the mucosa-associated lymphoid tissue.

[edit] Formation of lymph
Formation of interstitial fluid from blood

Blood supplies nutrients and important metabolites to the tissues, and collects back the waste products that they produce, which requires exchange of respective constituents between the blood and tissues. This exchange is not direct, however, and is effected through an intermediary called interstitial fluid or tissue fluid that the blood forms. Interstitial fluid (ISF) is the fluid that occupies the spaces between the cells and acts as their immediate environment. As the blood and the surrounding cells continually add and remove substances from the ISF, its composition keeps on changing. Water and solutes can freely pass (diffuse) between the ISF and blood, and thus both are in dynamic equilibrium with each other; exchange between the two fluids occurs across the walls of small blood vessels called capillaries.
Formation of lymph from interstitial fluid (labeled here as "Tissue fluid"). Note: how the tissue fluid is entering the blind ends of lymph capillaries (shown as deep green arrows)

ISF forms at the arterial (coming from the heart) end of the capillaries because of higher pressure of blood, and most of it returns to its venous ends and venules; the rest (10—20%) enters the lymph capillaries as lymph.[1] Thus, lymph when formed is a watery clear liquid with the same composition as the ISF. As it flows through the lymph nodes, however, it comes in contact with blood and tends to accumulate more cells (particularly lymphocytes) and proteins.[2]

The two primary lymph systems are the thymus gland and the bone marrow, where the immune cells form or mature. The secondary lymph system is made up of encapsulated and unencapsulated diffuse lymphoid tissue. The encapsulated tissue includes the spleen and the lymph nodes. The unencapsulated tissue includes the gut-associated lymphoid tissues and the tonsils.


[edit] Lymphoid tissue

Lymphoid tissue associated with the lymphatic system is concerned with immune functions in defending the body against the infections and spread of tumors. It consists of connective tissue with various types of white blood cells enmeshed in it, most numerous being the lymphocytes.

The lymphoid tissue may be primary, secondary, or tertiary depending upon the stage of lymphocyte development and maturation it is involved in. Primary (central) lymphoid tissues serve to generate mature virgin lymphocytes from immature progenitor cells. Secondary (peripheral) lymphoid tissues provide a place where lymphocytes can talk to each other; an environment for antigen focusing, where lymphocytes can 'study' an antigen and sharpen up the immune response by clonal expansion and affinity maturation; and provide a home for lymphocytes, where they can be available when they are needed.

The thymus and the bone marrow constitute the primary lymphoid tissues involved in the production and early selection of lymphocytes. Secondary lymphoid tissue provides the environment for the foreign or altered native molecules (antigens) to interact with the lymphocytes. It is exemplified by the lymph nodes, and the lymphoid follicles in tonsils, Peyer's patches, spleen, adenoids, skin, etc. that are associated with the mucosa-associated lymphoid tissue (MALT). The tertiary lymphoid tissue typically contains far fewer lymphocytes, and assumes an immune role only when challenged with antigens that result in inflammation. It achieves this by importing the lymphocytes from blood and lymph.[3]

[edit] Lymph nodes
For more details on this topic, see lymph node.
A lymph node showing afferent and efferent lymphatic vessels

A lymph node is an organized collection of lymphoid tissue, through which the lymph passes on its way to returning to the blood. Lymph nodes are located at intervals along the lymphatic system. Several afferent lymph vessels bring in lymph, which percolates through the substance of the lymph node, and is drained out by an efferent lymph vessel.

The substance of a lymph node consists of lymphoid follicles in the outer portion called the "cortex", which contains the lymphoid follicles, and an inner portion called "medulla", which is surrounded by the cortex on all sides except for a portion known as the "hilum". The hilum presents as a depression on the surface of the lymph node, which makes the otherwise spherical or ovoid lymph node bean-shaped. The efferent lymph vessel directly emerges from the lymph node here. The arteries and veins supplying the lymph node with blood enter and exit through the hilum.

Lymph follicles are a dense collection of lymphocytes, the number, size and configuration of which change in accordance with the functional state of the lymph node. For example, the follicles expand significantly upon encountering a foreign antigen. The selection of B cells occurs in the germinal center of the lymph nodes.

Lymph nodes are particularly numerous in the mediastinum in the chest, neck, pelvis, axilla (armpit), inguinal (groin) region, and in association with the blood vessels of the intestines.[1]

[edit] Lymphatics
lymphatic system

Tubular vessels transport back lymph to the blood ultimately replacing the volume lost from the blood during the formation of the interstitial fluid. These channels are the lymphatic channels or simply called lymphatics.[4]

[edit] General structure of Lymphatics

The general structure of lymphatics is based on that of blood vessels. There is an inner lining of single flattened cells composed of a type of epithelium that is called endothelium, and the cells are called endothelial cells. This layer functions to mechanically transport fluid and since the basement membrane on which it rests is discontinuous; it leaks easily.[5] The next layer is that of smooth muscles that are arranged in a circular fashion around the endothelium, which by shortening (contracting) or relaxing alter the diameter (caliber) of the lumen. The outermost layer is the adventitia that consists of fibrous tissue. The general structure described here is seen only in larger lymphatics; smaller lymphatics have fewer layers. The smallest vessels (lymphatic or lymph capillaries) lack both the muscular layer and the outer adventitia. As they proceed forward and in their course are joined by other capillaries, they grow larger and first take on an adventitia, and then smooth muscles.

The whole lymphatic conducting system broadly consists of two types of channels—the initial lymphatics, the prelymphatics or lymph capillaries that specialize in collection of the lymph from the ISF, and the larger lymph vessels that propel the lymph forward.

Unlike the cardiovascular system, the lymphatic system is not closed and has no central pump. Lymph movement occurs despite low pressure due to peristalsis (propulsion of the lymph due to alternate contraction and relaxation of smooth muscle), valves, and compression during contraction of adjacent skeletal muscle and arterial pulsation.[6]

[edit] Lymph capillaries
Propulsion of lymph through lymph vessel

The lymphatic circulation begins with blind ending (closed at one end) highly permeable superficial lymph capillaries, formed by endothelial cells with button-like junctions between them that allow fluid to pass through them when the interstitial pressure is sufficiently high.[7] These button-like junctions consist of protein filaments like platelet endothelial cell adhesion molecule-1 or (PECAM-1). A valve system in place here prevents the absorbed lymph from leaking back into the ISF. There is another system of semilunar (semi=half; lunar=related to the Moon) valves that prevents back-flow of lymph along the lumen of the vessel.[7] Lymph capillaries have many interconnections (anastomoses) between them and form a very fine network.[8]

Rhythmic contraction of the vessel walls through movements may also help draw fluid into the smallest lymphatic vessels, capillaries. If tissue fluid builds up the tissue will swell; this is called edema. As the circular path through the body's system continues, the fluid is then transported to progressively larger lymphatic vessels culminating in the right lymphatic duct (for lymph from the right upper body) and the thoracic duct (for the rest of the body); both ducts drain into the circulatory system at the right and left subclavian veins. The system collaborates with white blood cells in lymph nodes to protect the body from being infected by cancer cells, fungi, viruses or bacteria. This is known as a secondary circulatory system.

[edit] Lymph vessels

The lymph capillaries drain the lymph to larger contractile lymphatics, which have valves as well as smooth muscle walls. These are called the collecting lymphatics.[6] As the collecting lymph vessel accumulates lymph from more and more lymph capillaries in its course, it becomes larger and is called the afferent lymph vessel as it enters a lymph node. Here the lymph percolates through the lymph node tissue and is removed by the efferent lymph vessel. An efferent lymph vessel may directly drain into one of the (right or thoracic) lymph ducts, or may empty into another lymph node as its afferent lymph vessel.[8] Both the lymph ducts return the lymph to the blood stream by emptying into the subclavian veins

The functional unit of a lymph vessel is known as a lymphangion, which is the segment between two valves. Since it is contractile, depending upon the ratio of its length to its radius, it can act either like a contractile chamber propelling the fluid ahead, or as a resistance vessel tending to stop the lymph in its place.[9]

[edit] Function of the fatty acid transport system

Lymph vessels called lacteals are present in the lining of the gastrointestinal tract, predominantly in the small intestine. While most other nutrients absorbed by the small intestine are passed on to the portal venous system to drain, via the portal vein, into the liver for processing, fats (lipids) are passed on to the lymphatic system, to be transported to the blood circulation via the thoracic duct. The enriched lymph originating in the lymphatics of the small intestine is called chyle. As the blood circulates, fluid leaks out into the body tissues. This fluid is important because it carries food to the cells and waste back to the bloodstream. The nutrients that are released to the circulatory system are processed by the liver, having passed through the systemic circulation. The lymph system is a one-way system, transporting interstitial fluid back to blood.

[edit] Diseases of the lymphatic system

Lymphedema is the swelling caused by the accumulation of lymph fluid,[10] which may occur if the lymphatic system is damaged or has malformations. It usually affects the limbs, though face, neck and abdomen may also be affected. An estimated 170 million people develop lymphedema, which progresses in three stages:
Stage 1: Pressing the swollen limb leaves a pit that takes a while to fill back in. Because there is little fibrosis (hardening) it is often reversible. Elevation reduces swelling.
Stage 2: Pressure does not leave a pit. Elevation does not help. If left untreated, the limb becomes fibrotic.
Stage 3: This stage of lymphedema is often called elephantiasis. It is generally only in the legs after lymphedema that has gone long untreated. While treatment can help a little, it is not reversible.

Some common causes of swollen lymph nodes include infections, infectious mononucleosis, and cancer, e.g. Hodgkin's and non-Hodgkin's lymphoma, and metastasis of cancerous cells via the lymphatic system. In elephantiasis, infection of the lymphatic vessels cause a thickening of the skin and enlargement of underlying tissues, especially in the legs and genitals. It is most commonly caused by a parasitic disease known as lymphatic filariasis. Lymphangiosarcoma is a malignant soft tissue tumor (soft tissue sarcoma), whereas lymphangioma is a benign tumor occurring frequently in association with Turner syndrome. Lymphangioleiomyomatosis is a benign tumor of the smooth muscles of the lymphatics that occurs in the lungs.

[edit] Development of lymphatic tissue

Lymphatic tissues begin to develop by the end of the fifth week of embryonic life. Lymphatic vessels develop from lymph sacs that arise from developing veins, which are derived from mesoderm.

The first lymph sacs to appear are the paired jugular lymph sacs at the junction of the internal jugular and subclavian veins. From the jugular lymph sacs, lymphatic capillary plexuses spread to the thorax, upper limbs, neck and head. Some of the plexuses enlarge and form lymphatic vessels in their respective regions. Each jugular lymph sac retains at least one connection with its jugular vein, the left one developing into the superior portion of the thoracic duct.

The next lymph sac to appear is the unpaired retroperitoneal lymph sac at the root of the mesentery of the intestine. It develops from the primitive vena cava and mesonephric veins. Capillary plexuses and lymphatic vessels spread form the retroperitoneal lymph sac to the abdominal viscera and diaphragm. The sac establishes connections with the cisterna chyli but loses its connections with neighboring veins.

The last of the lymph sacs, the paired posterior lymph sacs, develop from the iliac veins. The posterior lymph sacs produce capillary plexuses and lymphatic vessels of the abdominal wall, pelvic region, and lower limbs. The posterior lymph sacs join the cisterna chyli and lose their connections with adjacent veins.

With the exception of the anterior part of the sac from which the cisterna chyli develops, all lymph sacs become invaded by mesenchymal cells and are converted into groups of lymph nodes.

The spleen develops from mesenchymal cells between layers of the dorsal mesentery of the stomach. The thymus arises as an outgrowth of the third pharyngeal pouch.

[edit] History

Hippocrates was one of the first persons to mention the lymphatic system in fifth century BC. In his work "On Joints," he briefly mentioned the lymph nodes in one sentence. Rufus of Ephesus, a Roman physician, identified the axillary, inguinal and mesenteric lymph nodes as well as the thymus during the first to second century AD.[11] The first mention of lymphatic vessels was in 3rd century BC by Herophilus, a Greek anatomist living in Alexandria, who incorrectly concluded that the "absorptive veins of the lymphatics", by which he meant the lacteals (lymph vessels of the intestines), drained into the hepatic portal veins, and thus into the liver.[11] Findings of Ruphus and Herophilus findings were further propagated by the Greek physician Galen, who described the lacteals and mesenteric lymph nodes which he observed in his dissection of apes and pigs in the second century A.D.[11][12]

Until the seventeenth century, ideas of Galen were most prevalent. Accordingly, it was believed that the blood was produced by the liver from chyle contaminated with ailments by the intestine and stomach, to which various spirits were added by other organs, and that this blood was consumed by all the organs of the body. This theory required that the blood be consumed and produced many times over. His ideas had remained unchallenged until the seventeenth century, and even then were defended by some physicians.[12]
Olaus Rudbeck in 1696

In the mid 16th century Gabriel Fallopius (discoverer of the Fallopian Tubes) described what are now known as the lacteals as "coursing over the intestines full of yellow matter."[11] In about 1563 Bartolomeo Eustachi, a professor of anatomy, described the thoracic duct in horses as vena alba thoracis.[11] The next breakthrough came when in 1622 a physician, Gasparo Aselli, identified lymphatic vessels of the intestines in dogs and termed them venae alba et lacteae, which is now known as simply the lacteals. The lacteals were termed the fourth kind of vessels (the other three being the artery, vein and nerve, which was then believed to be a type of vessel), and disproved Galen's one idea wrong: that chyle was carried by the veins. But, he still believed that the lacteals carried the chyle to the liver (as taught by Galen).[13] He also identified the thoracic duct but failed to notice its connection with the lacteals.[11] This connection was established by Jean Pecquet in the 1651, who found a white fluid mixing with blood in a dog's heart. He suspected that fluid to be chyle as its flow increased when abdominal pressure was applied. He traced this fluid to the thoracic duct, which he then followed to a chyle-filled sac he called the chyli receptaculum, which is now known as the cisternae chyli; further investigations led him to find that lacteals' contents enter the venous system via the thoracic duct.[11][13] Thus, it was proven convincingly that the lacteals did not terminate in the liver, thus disproving Galen's second idea that the chyle flowed to the liver.[13] Johann Veslingius drew the earliest sketches of the lacteals in humans in 1647.[12]
Thomas Bartholin

The idea that blood recirculates through the body rather than being produced anew by the liver and the heart was first accepted as a result of works of William Harvey—a work he published in 1628. In 1652, Olaus Rudbeck (1630–1702), a Swede, discovered certain transparent vessels in the liver that contained clear fluid (and not white), and thus named them hepatico-aqueous vessels. He also learned that they emptied into the thoracic duct, and that they had valves.[13] He announced his findings in the court of Queen Christina of Sweden, but did not publish his findings for a year,[14] and in the interim similar findings were published by Thomas Bartholin, who additionally published that such vessels are present everywhere in the body, and not just the liver. He is also the one to have named them "lymphatic vessels".[13] This had resulted in a bitter dispute between one of Bartholin's pupils, Martin Bogdan,[15] and Rudbeck, whom he accused of plagiarism.[14]

[edit] See also

replication

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

recombination

Genetic recombination is the process by which a strand of genetic material (usually DNA; but can also be RNA) is broken and then joined to a different DNA molecule. In eukaryotes recombination commonly occurs during meiosis as chromosomal crossover between paired chromosomes. This process leads to offspring having different combinations of genes from their parents and can produce new chimeric alleles. In evolutionary biology this shuffling of genes is thought to have many advantages, including allowing asexually reproducing organisms to avoid Muller's ratchet.

In molecular biology "recombination" can also refer to artificial and deliberate recombination of disparate pieces of DNA, often from different organisms, creating what is called recombinant DNA.

Enzymes called recombinases catalyze natural recombination reactions. RecA, the recombinase found in E. coli, is responsible for the repair of DNA double strand breaks (DSBs). In yeast and other eukaryotic organisms there are two recombinases required for repairing DSBs. The RAD51 protein is required for mitotic and meiotic recombination and the DMC1 protein is specific to meiotic recombination.

replication bubble

Replication bubble
Once the polymerases have opened the molecule, an area known as the replication bubble forms (always initiated at a certain set of nucleotides, the origin of replication).

meselson and stahl experiment

Meselson-Stahl experiment
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A summary of the three postulated methods of DNA synthesisThe Meselson-Stahl experiment was an experiment by Matthew Meselson and Franklin Stahl which demonstrated that DNA replication was semiconservative. Semiconservative replication means that when the double stranded DNA helix was replicated, each of the two double stranded DNA helices consisted of one strand coming from the original helix and one newly synthesized.

Nitrogen is a major constituent of DNA. 14N is by far the most abundant isotope of nitrogen, but DNA with the heavier 15N isotope is also viable. The 15N isotope is not radioactive, only heavier than common nitrogen.

E. coli were grown for several generations in a medium with 15N. When DNA is extracted from these cells and centrifuged on a salt density gradient, the DNA separates out at the point at which its density equals that of the salt solution. The DNA of the resulting cells had a higher density (was heavier). After that, E. coli cells with only 15N in their DNA were put back into a 14N medium and were allowed to divide only once. DNA was then extracted from a cell and was compared to DNA from 14N DNA and 15N DNA. It was found to have close to the intermediate density. Since conservative replication would result in equal amounts of DNA of the higher and lower densities (but no DNA of an intermediate density), conservative replication was excluded. However, this result was consistent with both semiconservative and dispersive replication. Semiconservative replication would result in double-stranded DNA with one strand of 15N DNA, and one of 14N DNA, while dispersive replication would result in double-stranded DNA with both strands having mixtures of 15N and 14N DNA, either of which would have appeared as DNA of an intermediate density.

DNA was then extracted from cells which had been grown for several generations in a 15N medium, followed by two divisions in a 14N medium. DNA from these cells was found to consist of equal amounts of two different densities, one corresponding to the intermediate density of DNA of cells grown for only one division in 14N medium, the other corresponding to cells grown exclusively in 14N medium. This was inconsistent with dispersive replication, which would have resulted in a single density, lower than the intermediate density of the one-generation cells, but still higher than cells grown only in 14N DNA medium, as the original 15N DNA would have been split evenly among all DNA strands. The result was consistent with semiconservative replication, in that half of the second-generation cells would have one strand of the original 15N DNA along with one of 14N DNA, accounting for the DNA of intermediate density, while the DNA in the other half of the cells would consist entirely of 14N DNA--one synthesized in the first division, and the other in the second division. This discovery was hugely important for the development of biology and is a majory aid to the treatment of disease (e.g. cancer).

replication types

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.


[edit] 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.


[edit] 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 hemi-methylated DNA

replication fork

The replication fork is a structure that forms within the nucleus during DNA replication. It is created by helicases, which break the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching "prongs", each one made up of a single strand of DNA, that are called the leading and lagging strands. DNA polymerase creates new partners for the two strands by adding nucleotides