Replication Forks and Origins of Replication

In the cell, both strands of the DNA duplex are replicated at the same time. The first step in DNA replication is the separation of the two DNA strands that make up the helix that is to be copied to make two template DNAs. DNA Helicase untwists the helix at locations called replication origins. The junction between the newly separated template strands and the unreplicated duplex DNA is known as the replication fork.

The replication fork moves down the DNA strand, usually from an internal location to the strand’s end (unreplicated DNA). The result is that every replication fork has a twin replication fork, moving in the opposite direction from that same internal location to the strand’s opposite end.

Single-stranded binding proteins (SSB) work with helicase to keep the parental DNA helix unwound. It works by coating to stabilize the unwound strands with rigid subunits of SSB that keep the strands from snapping back together in a helix. Binding of one SSB promotes the binding of another SSB to the immediately adjacent ssDNA. This is called cooperative binding.The SSB subunits coat the single-strands of DNA in a way as not to cover the bases, allowing the DNA to remain available for base-pairing with the newly synthesized daughter strands.

As the strands of DNA are seperated at the replication fork, the duplex DNA infront of the fork becomes increasingly positively supercoiled. This accumulation of super is the result of DNA helicase eliminating the base parts between the two strands. If there is no mechanism to relieve the accumulation of these supercoils, the replicating machinery would grind to a halt in the face of mounting pressure. These super coils produced by the DNA unwinding at the replication fork is removed by Toposiomerases.

Because of the antiparallel nature of DNA and because DNA is only sythesized by elongating 3′ end, daughter strands synthesize through different methods, one adding nucleotides one by one in the direction of the replication fork, the other able to add nucleotides only in chunks. The first strand, which replicates nucleotides one by one (continuously) is called the leading strand; the other strand, which replicates in chunks (discontinuous fashion), is called the lagging strand.

Leading strand DNA polymerase can replicate its template as soon as it is exposed, synthesis of the lagging strand must wait for movement of the replication fork to expose a substantial length of new lagging strand is exposed.

The lagging strand replicates in small segments, called Okazaki fragments. These fragments are stretches of 100 to 200 nucleotides in humans (1000 to 2000 in bacteria) that are synthesized in the 5′ to 3′ direction away from the replication fork. Yet while each individual segment is replicated away from the replication fork, each subsequent Okazaki fragment is replicated more closely to the receding replication fork than the fragment before. These fragments are then stitched together by DNA ligase, creating a continuous strand. This type of replication is called discontinuous.

The initiation of New strand of DNA requires an RNA Primer with free 3’OH. DNA polymerase cannot initiate a new DNA strand them selves. To accomplish this the cell take advantage of the ability of RNA polymerase to do what DNA polymerases cannot: initiate new RNA strand. Primase is a special RNA polymerase dedicated to making short, RNA primers on an ssDNA template. These primers are subsequently extended by DNA Polymerase.

Although both the leading and lagging strands require primase to initiate DNA synthesis, the frequency of primase function on the two strands is dramatically different. Leading strand requires only a single RNA primer. In contrast, the discontinuous synthesis of the lagging strand means that new primers are needed for each Okazaki fragments.

Primase does not require a specific DNA sequence to initiate the synthesize of new RNA primer. Instead primase is activated only when it is associated with other DNA replication protein such as DNA helicase. Once activated, the primase synthesize a RNA primer using the most recently exposed lagging strand template regardless of sequence.

To complete the DNA replication, the RNA primase used for the initiation must be removed and replaced by DNA. to replace the RNA primers with DNA, an enzyme called RNase H recognize and removes most of the each RNA primer. This enzyme specifically degrades RNA that is base paired with DNA. RNase H removes all of the RNA primer except the ribonucleotide directly linked to the DNA end. This is because RNase H can only cleave bonds between tow ribonucleotides.The final ribonuceotide is removed by an a exonuclease, that degrages RNA and DNA from there 5′ end.

Removal of RNA leaves a gap in the double-stranded DNA. DNA polymerase fill the gap until every nucleotide is base-paired, leaving a DNA molecule that is complete except for the break in the backbone bvetween ythe 3’Oh and 5′ phosphate of the repaired strand. This nick in the DNA can be repaired by an enzymes called DNA ligase. Only after all RNA primase are replaced and asscociated nicks are sealed, DNA synthesis is completed.

Mechanism of DNA Polymerase

The synthesis of DNA is catalyzed by an enzyme called DNA Polymerase.  Unlike most enzymes, which have an active site dedicated to a single reaction, DNA polymerase uses a single active site  to catalyze addition of any of the four deoxynucleotide triphosphates. Polymerase shows kinetic selectivity, in which an enzyme favors catalysis using one of several possible substrates by dramatically increasing the rate of bond formation only when the correct substrate is present. Only when the correct pair is formed the catalysis occurs. Incorrect base pairing leads to dramatically lower rate of nucleotide addition due to the catalytically unfavorable alignment of these substrates. DNA polymerase show an impressive ability to discriminate between ribo- and deoxyribonucleoside phosphates. This ability is mediated by steric exclusion of rNTPs from the DNA polymerase active site, because in DNA polymerase the nucleotide binding pocket is too small to allow the presence of a 2’OH on the incoming nucleotide.

DNA Polymerase Structure

DNA Polymerase resemble a right hand that partially grips the primer: Template junction. Based on anatomy of the hand, there are three domains in the polymerase:

  • Palm: The palm domain is composed of a B Sheet and is the active site of DNA Polymerase. This region of DNA polymerase binds two divalent metal ions (Mg2+ or Zn2+) that alter the chemical environment around the correctly base-paired dNTP and the 3’OH of the primer. One metal ion reduces the affinity of the 3’OH for its hydrogen. This generates a 3’O- that is primed for the nucleophilic attack of the alpha-phosphate of the incoming dNTP. The secong metal ion coordinates the negative charges of the Beta and Gamma phosphates of the dNTP and stabilizes the pyrophosphate produced by joining the primer and the incoming nucleotide. The palm domain also monitors the accuracy of base-pairing for the most recently added dNTP.
  • Fingers: once the correct base-pair is formed, the finger domain moves to enclose the dNTP. this close form of the polymerase hand stimulates catalysis by moving the incoming nucleotide in close contact with the catalytic metal ions.
  • Thumb: Thumb interacts with the DNA that has been most recently synthesized. this serves two purpose: (1) It maintains the correct position of the primer and the active site, (2) helps to maintain a strong association between the DNA polymerase and its substrate, which contribute to the processive nature of DNA polymerase.

Features of DNA Polymerase:

  • High Processivity: Catalysis by DNA polymerase is rapid, are capable of adding as many as 1000 nucleotide per second to a primer strand. In case of DNA polymerase, the degree of processivity is defined as the number of nucleotides added each time the enzyme binds a primer:template junction.
  • Multiple substrates: In processive enzyme each time the enzyme binds a primer:template junction multiple substrates, dNTP is added to the primer. In contrast with no processive enzyme adds a single dNTP to primer and then is released from the primer:template junction.

Increased processivity is facilitated by the ability of DNA polymerase to slide along the DNA Template. DNA polymerase interacts tightly with much of the double-stranded portion of the DNA in a sequence non specific manner. These interactions include electrostatic interactions between the phosphate backbone and the thumb domain, and interaction between the minor groove of the DNA and the palm domain. Further increase in processivity are achived through the interactions between the DNA polymerase and the “Sliding clamp” protein.

  • Exonuclease Activity: Occasional flickering of the bases into the ‘wrong’ tautomeric form. This alternate form of bases allow incorrect base pairs to be correctly positioned for catalysis. Exonucleases proofread newly synthesized DNA. These type of exonuclease is called proofreading exonuclease. Theses exonucleases are capable of degrading DNA starting from the 3′ DNA end, that is the from the growing end of the new DNA strand and remove the incorrect nucleotide.

Mispaired DNA alters the geometry of the 3′ OH and the incoming nucleotide due to poor interactions with the palm region. This altered geometry reduces the rate of nucleotide addition and increase the rate of proofreading exonuclease activity. proofreading can occur without releasing the DNA from the polymerase. When a mismatched pair is detected, the primer:template junction slides away from the DNA polymerase active site and into the exonuclease site. after the incorrect nucleotide is removed the correctly paired primer:template junction slides back ingto the active site of polymerase and DNA synthesis continues.

The DNA polymerase performing synthesis and editing

  • Nucleotides are added in to both templates in replication fork at the same time.
  • Primer is required. DNA polymerase require primer to initiate DNA Polymerase.

Specialization of DNA Polymerase

DNA polymerases are specialized for different roles in the cell. The central role of DNA polymerases in the efficient and accurate replication of the genome requires that cell have multiple specialized DNA polymerases.

DNA polymerases in Prokaryotes:

1. DNA polymerase II (pol II) is a minor DNA polymerase in E. соli.

  • Function. Pol II may be involved in some DNA repair processes, but E соli mutants lacking this enzyme show no replication or growth deficiencies.
  • Structure. Pol II is a single polypeptide.
  • Other enzymatic activities. Pol II has proofreading (3′ to 5′ exonuclease) activity but lacks excision-repair (5′ to 3′ exonuclease) activity.

2. DNA polymerase III (pol III) is the primary DNA polymerase involved in cellular replication.

  • Function. Pol III catalyzes leading and lagging strand synthesis.
  • Structure. Pol III is structurally complex. The pol III core enzyme, made of three different polypeptides, was first isolated. Gentle purification schemes and careful reconstitution showed that the active cellular form of pol HI, called pol III holoenzyme, is made of 10 subunit polypeptides. The subunits are organized into an asymmetric dimeric structure with two catalytic centers. This is important in understanding how it catalyzes both leading and lagging strand syntheses at the replication fork.
  • Other enzymatic activities. Pol III holoenzyme has proofreading (3′ to 5′ exonuclease) activity but no excision-repair (5′ to 3′ exonuclease) activity. The two α subunits both contain the DNA polymerase activity, and the two ε subunits both contain the 3′ to 5′ exonuclease activity. The two subunits form a dimer that circles around the DNA. This β2 “DNA clamp” enables the pol III holoenzyme to stay bound to the DNA and facilitates its high rate of replication.

3. DNA polymerase I (pol I) was the first DNA polymerase discovered.

  • Function. Pol I functions in the replication of DNA and in the repair of damaged DNA.
  • Structure. Pol I is a single polypeptide.
  • Other enzymatic activities. Pol I has two enzymatic activities—besides DNA polymerase activity—that are important to its cellular function.
    • (1) Proofreading. Pol I does not typically add a nucleotide to the growing DNA chain that cannot properly base pair with the template strand. If a mismatched nucleotide is added, the enzyme halts polymerization. A 3′- to 5′-exonuclease activity removes the mismatched nucleotide, and polymerization resumes. This activity is called proofreading, and it assures the high-level fidelity of replication that is a desirable trait of genetic material.
    •  (2) Excision-repair. Pol I has a 5′ to 3′ exonuclease activity, called excision-repair activity, that can hydrolytically remove a segment of DNA from the 5′ end of a strand of duplex DNA.
      • (a) From 1-10 nucleotide segments of DNA can be removed simultaneously.
      • (b) This activity is essential for the removal of primers in DNA replication.
      • (c) This activity is essential for the repair of damaged DNA.
    • (3) Nick-translation. A nick is a break in a phosphodiester bond of one strand of DNA in a double helix. A nick leaves a free 3′-hydroxyl and a free 5′-phosphate. Pol I can function at nicks as an exonuclease and a polymerase at the same time. As the 5′-phosphate nucleotide is removed or displaced by pol I, it is replaced with the polymerase activity of pol I. Pol I cannot reseal the nick; therefore, the nick is moved, or translated, along the DNA in the direction of synthesis.

DNA polymerases in Eukaryotes:

1. DNA polymerase alpha /primase

  • Function. DNA polymerase alpha is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme.
  • Structure. Pol alpha contain 4 subunit. (two subunit DNA Pol alpha and a two-subunit primase)

2. DNA polymerase beta

  • Function. Base excision repair. The role of DNA polymerase beta in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase delta appears to fulfill that function.
  • Structure. Pol beta contain 1 subunit.

3. DNA polymerase gamma

  • Function. Mitrochondrial DNA replication and repair.
  • Structure. contain 3 subunit.

4. DNA polymerase Delta

  • Function. DNA polymerase delta functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase zeta, for mutagenesis.
  • Structure. contain 2-3 subunits.

5. DNA polymerase Epsilon

  • Function. The function of DNA polymerase epsilon in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase delta or epsilon suffices for the repair of UV-induced damage.
  • Structure. contain 4 subunits.

During Replication, due to relatively low processivity , DNA pol alpha/primase is replaced by the highly processive DNA polymerase Delta and Epsilon by the process called polymerase switching. The majority of other DNA polymerase in Eukaryotes are involved in DNA repair.

DNA Replication: Introduction

DNA replication is the process of producing two identical replicas from one original DNA molecule. This biological process occurs in all living organisms and is the basis for biological inheritance. DNA is made up of two strands and each strand of the original DNA molecule serves as template for the production of the complementary strand, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.

For the DNA synthesis two key substrate is required.

Substrate: The four deoxynucleoside triphosphates (dNTPs)—deoxyadenosine tri- phosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphos-phate (dCTP), and deoxythymidine triphosphate (dTTP)—are needed as substrates for DNA synthesis.

Primer:template junction: second substrate for the DNA Synthesis is a particular arrangement of ssDNA and dsDNA called primer:template junction.

  • Template. DNA replication cannot occur without a template. A template is required to direct the addition of the appropriate complementary deoxynucleotide to the newly synthesized DNA strand. In semiconservative replication, each strand of parental DNA serves as a template. Then, each template strand and its newly synthesized complementary strand serve as the DNA in daughter cells.
  • Primer. DNA synthesis cannot start without a primer, which prepares the template strand for the addition of nucleotides. The primer must also have an exposed 3’OH because new nucleotides are added to the 3′ end of a primer that is properly base paired to the template strand of DNA, new synthesis is said to occur in a 5′ to 3′ direction.

The DNA synthesis that occurs during the process of replication is catalyzed by enzymes called DNA-dependent DNA polymerases. These enzymes depend on DNA to the extent that they require a DNA template. They are more commonly called DNA polymerases.

DNA and its structural Properties.

DNA is the prime genetic molecule carrying all the hereditary information within chromosomes.

DNA Structure.

The most important feature of DNA is that it is usually composed of two polynucleotide chains twisted around each other in the form of right handed double helix. These two strands are complementary to each other.

However, since each strand has a both a free 5′ hydroxyl group at one end and a free 3′ hydroxyl group at the other, each strands has a polarity or directionality. The polarity of these two strands of macromolecule are in opposite directions, and thus DNA is described as an ‘anti-parallel‘ structure.

These macro molecule structures are composed of regular repeating polymers formed from nucleotides. These are the basic building blocks of nucleic acids. The nucleotides are derived from nucleosides that are composed of two elements; a five-membered pentose sugar, known as 2′-deoxyribose and a nitrogenous base. The sugar is called 2′-deoxyribose because there is no hydroxyl at position 2′. (just two hydrogen). The carbon atom of the sugar are designated ‘prime’ (1′,2′,3′,…5′) to distinguish them from the carbon atoms of nitrogenous bases.

A nucleotide or nucleoside phosphate is fromed by the attachment of a phosphate to the 5′ position of a nucloside by an ester linkage. Such nuclotides can be joined together by the formation of a second ester bond by reaction between the terminal phosphate group of one nucleotide and the 3′ hydroxyl of another, thus generating a 5′ to 3′ phophodiester bond between adjacent sugars; this process can be repeated indefinitely to give long polynucleotide molecule.

The bases in DNA falls in to two classes; purines and pyrimidines. The purines are adenine (A) and Guanine (G) and the pyrimidines are cytosin (C) and thymine (T). The purines are derived from the double-ringed structure but with different groups attached. The pyrimidines are single-ringed structures. The bases are attached to the deoxyribose by Glucosidic linkages at N1 of the pyrimidines or N9 of the purines.

The sequence of bases at one strand is complementary to that in the other. A purine base attached to a sugar residue on one strand is always hydrogen bonded to a sugar residue on the other strand. Adenine always pairs with thymine, via two hydrogen bonds, and guanine always pairs with cytosin by three hydrogen bonds. This is called the specificity of base pairing.

These bases exists in two alternative tautomeric states. The nitrogen atoms attached to the purines and pyrimidine rings are in amino form in the predominant state and only rarely assume the imino configuration. Likewise, the oxygen atoms attached to the guanine and thymine normally have the keto form and only rarely take on the enol configuration.

The high degree of thermodynamic stability of DNA double helices result in part from the large number of hydrogen bonds between base-pairs and in part from the hydrophobic bonding (or “staking force“) between staked base-pairs.

An organic molecule in aqueous solution has all its hydrogen bonding properties satisfied with by water molecules, that comes on and off very rapidly. This means for every hydrogen bond formed requires breaking of H-bond with water molecule that was there before the base pair formed. However, when the polynucleotide chains are seperated, water molecules are lined up on the bases. when strands come together in the double helix, the water molecules are displaced from the bases. This creates disorder and increase entropy, thereby stabilizing the double helix.

DNA double helix is also stabilized by the stacking interactions between the bases. The bases are flat, relatively insoluble molecules, and they stack above each other roughly perpendicular to direction of double helix. Electron cloud interaction (Pi-Pi) between bases in the helical stacks contribute significantly to the stability of the double helix.

Hydrogen bonding is also important for the specificity of the base pairing. Suppose we tried to pair an Adenine with a Cytosine, then they have a hydrogen acceptor (N3 of cytosine) lying opposite a H-Bond acceptor (N1 of Adenine) with no room to put a water molecule in between to satisfy the two acceptors. And like wise two hydrogen bond donor opposite each other. Thus an A:C base pair is unstable in forming H-Bond.

 

 

 

 

 

Mechanism of oncogenic viruses and Prevention.

How do virus causes cancer.

Most virus-induced cancers develop after a long period of persistant infection with an oncogenic virus. (for T cell leukaemia around 60 years).

Although many humans are persistently infected by the virus that are potentially oncogenic, only small percentages develop virus-linked cancers. So it is clear that virus alone do not cause cancer. Other factors are involved:

  • Complex interplay between the state of the host.
  • Environmental factors to which the host is exposed.
  • Host genetic factors.
  • Cellular changes induced by the virus.
  • Virus proteins synthesized during persistent infections play roles in the conversion of normal to cancer cells. 

 

Mechanisms Involved:

(a) ‘Delibrate’ interference with control of the cell cycle.

Many cells in the animal body grow and divide either slowly or not at all; the latter are arrested in the G1 phase of the cell cycle and are said to be in the G0 state. The control of the cell cycle is mediated by many proteins; two that play key role in humans are p53 and retinoblastoma protein (pRb).

Virus proteins that can interfere with the contol of the cell cycle include the HPV early proteins E6 and E7. Papillomavirus have small genomes about 8kbp and require the cell’s DNA synthesizing functions that are available in the S phase of the cell cycle. E6 and E7 make these functions available by localizing to the nucleus and binding to cell proteins.

E6 binds to p53, promoting its degradation, and E7 binds to pRb-E2F complexes causing their dissociation. E2F is an elongation factor which enhance DNA synthesis. As long as pRb is bound to E2F, no DNA syntheis will occur. The outcomes of this dissociation makes E2F free from pRb-E2F complexe and this transition will make the cell to move into S phase synthesizing viral DNA.

(b) ‘Accidental’ activation of cell genes.

Some virus proteins are able to bind to cell proteins that may not be the intended targets and may trigger events that are of no value to the virus, but may be harmful to host. 

A virus protein might inadvertently push a cell towards a cancerous state by activating a cell gene that is switched off, or by enhancing the rate of transcription of a gene that is being expressed at a low level. 

For example: Tax protein of HTLV-1, it activates the expression of its gene known as tax. this tax may interact with cell genes and some of proto-oncogenes (eg:c-myc) can be activated or their may be a boost in the expression resulting in cancer. 

 

(c) Retroviral Oncogenes 

Some retroviruses have the ability to cause cancer because of the presence in the viral genome of an oncogene. The retroviral genes were drived from cell proto-oncogenes and are closely related to them. 

When a proto-oncogene is mutated or deleted or aberrantly expressed it becomes an oncogene. The oncogene-carrying retroviruses have the ability to induce rapid formation of tumors, usually 1-6 weeks post infection.

Interaction between cell proteins and proteins produced by oncogenic viruses can lead to breakdown of immune defenses that may allow development of cancer,

1st mechanism: RNA by Reverse transcriptase is converted in to DNA which gets integrated into host genome. If Viral DNA gets integrated near proto-oncogene it gets activated and gives hyper expression and result into oncogene and then cancer.

2nd mechanism: It posses a viral oncogenes. Viral DNA which contain V-onc genes get integrated into host genome resulting in cancer. 

 

Prevention of Viral induced Cancer: 

  • prevent transmission of viruses.
  • strategies aimed at reducing the risk of transmission 
  • vaccination
  • Attempted elimination from the body of persistent infections with oncogenic viruses.

Hepatocellular carcinoma

Hepatocellular carcinoma (HCC, also called malignant hepatoma) is the most common type of liver cancer. Most cases of HCC are secondary to either a viral hepatitis infection or cirrhosis (alcoholism being the most common cause of hepatic cirrhosis).

In HCC number of factors are implicated as causative agents including consuption of mould toxins in food, and two viruses: Hepatitis B virus (HBV) and Hepatitis C virus (HCV).

HBV is the most significant agent. HBV DNA is integrated into genome, and in most cases the virus DNA has undergone rearrangements, including deletions.

When HBV genome is integrated into human chromosomes, possible out comes are as follows: activates oncognes; cayses chromosome instability and integration disrupts, which all leads to tumor cell.

 

Human T cell leukemia/lymphotropic virus type 1 (HTLV-1)

Adult T-cell leukemia/lymphoma (ATLL) is a rare and  aggressive T-cell lymphoma that is linked to infection by the human T-cell lymphotropic virus 1 (HTLV-1). Human
T-cell lymphotropic virus 1 is a retrovirus—meaning that  these viruses do not contain gentic material made of DNA, but instead carry RNA.

These viruses selectively infect only T-cells. Only about 2 percent to 5 percent of patients infected with the HTLV-1 virus will develop ATLL. Currently, physicians have no way of telling which infected patients will develop the lymphoma.

The HTLV-1 virus is in the same class of virus as the HIV/ AIDS virus and is endemic to certain parts of the world such as Japan, the Caribbean, South and Central America, West Africa and the southeastern United States. The HTLV-1 virus is believed to be transmitted through sexual contact,  exposure to contaminated blood from either a blood transfusion or used needles and can be passed from mother to child through breastfeeding.

Kaposi’s sarcoma-associated herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the eighth human herpesvirus. This virus causesKaposi’s sarcoma, a cancer commonly occurring in AIDS patients, as well as primary effusion lymphoma and some types of multicentric Castleman’s disease. It is one of seven currently known human cancer viruses, or oncoviruses.

 

Structure and Genome

KSHV is a herpesvirus, and is a large double-stranded DNA virus with a protein covering that packages its nucleic acids, called the capsid, which is then surrounded by an amorphous protein layer called the tegument, and finally enclosed in a lipid envelope derived in part from the cell membrane. KSHV has a genome which is approximately 165,000 nucleic acid bases in length. It is a rhadinovirus, and is remarkable since it has stolen numerous genes from host cells including genes that encode for complement-binding protein, IL-6, BCL-2, cyclin-D, a G protein-coupled receptor, interferon regulatory factor and Flice inhibitory protein (FLIP), as well as DNA synthesis proteins including dihydrofolate reductase, thymidine kinase, thymidylate synthetase, DNA polymerase and many others.

History

Kaposi’s sarcoma was first described in the 19th century as a rare skin cancer that effected elderly men in the Maditteranean region. In 1994 it was discovered that the tumor cells contain the DNA of a new herpesvirus. The virus was subsequently isolated and was named Kaposi’s sarcoma-associated herpesvirus (KSHV).

 

Epidemiology

It is transmitted through sexsual contact and via organ transplant. Healthy individuals can be infected with the virus and show no signs or symptoms, due to the immune system’s ability to keep the infection in check. Kaposi’s sarcoma occurs when someone who has been infected with KSHV becomes immunocompromised due to AIDS, medical treatment or very rarely aging.

Its tumor cells are derived from endothelial cells. Two other human cancers linking with KSHV is : primary effusion lymphomas and Multicentric Castleman’s disease. Both are tumor derived from B cells,

Epstein – Barr Virus

The Epstein–Barr virus (EBV), also called human herpesvirus 4 (HHV-4), is a virus of the herpes family, and is one of the most common viruses in humans.

Structure and genome

The virus is approximately 122 nm to 180 nm in diameter and is composed of a double helix of DNA wrapped in a protein capsid. The capsid is surrounded by a tegument made of protein, which in turn is surrounded by a envelope made from lipids.The EBV DNA is about 192,000 base pairs long and contains about 85 genes.The viral envelope contains glycoproteins, which are essential to infection of the host cell.

 

Replication

It infects primarily lymphocytes and epithelial cells. In lymphocytes, the infection is usually non-productive, while virus is shed (produce infection) from infected epithelial cells.

EBV is casually associated with:

  • Burkitt’s Lymphoma in the tropics.
  • Nasopharyngeal cancer, particularly in china and south Asia, where certain diets acts as co-carcinogens.
  • B cell lymphomas in Immune suppressed individuals (as in organ transplantation or HIV).
  • Hodgkin’s lymphoma (in 40% of affected patients).
  • X-linked lymphoproliferative Disease (Duncan’s syndrome).
  • Non-Hodgkin’s lymphoma in AIDS.

Burkitt’s lymphoma is a B cell tumor that occurs with a high frequency in children in central Africa. The Epstein–Barr virus is named after Michael Anthony Epstein, a professor emeritus at the University of Bristol, and Yvonne Barr (born 1932 in London), a 1966 Ph.D graduate from theUniversity of London, who together discovered and documented the virus, So was named Esptein – Barr Virus.

A constant abnormality in BL tumor cells is a chromosomal rearrangement that results in the c-myc gene being placed next to an enhancer of an immunoglobulin gene. This results in the expression of c-myc at abnormally high levels. 

Phage Display

Phage Display Cycle

One of the laboratory techniques employed in studying different protein interactions is Phage Display. With this in vitro screening method, protein ligands and macromolecules can be easily identified and interactions between protein and protein, peptide and protein, & DNA and protein can be studied further.

History of Phage Display

The first described instance of Phage Display occurred in 1985, when George P. Smith fused a peptide with a gene III from a filamentous phage. He filed a patent detailing the process of generating phage display libraries in the same year. Eventually, further development of Phage Display technology led by different groups from the MRC Laboratory of Molecular Biology, as well as from The Scripps Research Institute, led to the possibility of displaying proteins for the purpose of therapeutic protein engineering. The technique has been continuously improved to screen and amplify huge collections of proteins showing the connection of phenotype and genotype better.

Structure

A filamentous phage has a diameter of around 6.5 nanometers, with a length that depends on the size of its genome. It comes from a huge family of bacterial viruses that also infect other forms of bacteria. It contains a small genome with an intergenic region containing the necessary sequences for the replication and encapsidation of DNA.

A phage particle consists of five coat proteins. The particle has a hollow tube that houses so many copies of the primary coat protein. There are also binding interactions between the adjacent subunits’ hydrophobic midsections. One end of the particle is blunt, and the other is sharp. The blunt end contains plenty of copies of the two tiniest ribosomally translated proteins, while the sharp end contains around only 5 copies of the pIII and pVI genes, which are necessary for the detachment of the phage from the cell membrane.

How it works

Phage Display is a method wherein a library of phage particles that express very diverse peptides is generated. The objective is to choose those that will bind a desired target; the target can be a protein, a peptide, or a piece of DNA.

The most often used vector to build a random peptide display is the filamentous phage M13. In this display, the DNA which encodes the peptide or protein of interest is integrated into the pIII or pVI gene. To make sure that the fragments are completely inserted into the three possible reading frames, multiple cloning sites are sometimes employed, allowing the proper translation of the cDNA in its correct frame. The DNA hybrid and the phage gene are then put inside E. coli bacterial cells. Examples of these bacterial cells include XL1-Blue E. coli, SS320, TG1, and ER2738. The peptide or protein of interest is eventually expressed in either the minor or major coat protein

If another kind of vector is used, for example, a phagemid vector or simplified display construct vector, a helper phage must infect the E. coli cells; otherwise, the phage particles will not be separated from the E. coli cells. A helper phage activates the packaging of the phage DNA and assembles the mature virions with their corresponding protein fragments, which are included in the outer coating of the minor or major protein coat.

The generated phage library is then screened by addition into a microtiter plate containing immobilised target proteins or DNA sequences. Phages displaying a protein that bind to one target will remain, while the other phages can be discarded through washing. The remaining phage particles can be used to multiply the phage by infecting them into bacteria, thus increasing the diversity of the peptide display library.

Applications

The fast isolation of particular ligands through phage display has a wide variety of applications like epitope mapping, analyzing different protein interactions, vaccine development, drug design, and therapeutic target validation. Phage display is also used to pick inhibitors for the active and allosteric sites of G-protein binding modulatory peptides, enzymes, and receptor antagonists and agonists.

Determining the proper protein partners can be useful to identify the functions of various proteins. For drug discovery and design, Phage Display is employed in protein engineering or in vitro protein evolution. Therapeutic targeting with phage display is also primarily used to diagnose and determine tumour antigens, which is useful for cancer research.

Antibody Phage Display significantly improved the discovery and development of antibody drugs. Phage display for antibody libraries paved the way for rapid vaccine design and therapy. These libraries are used to learn more about the human immune system and to create human antibodies in vitro with the use of diverse synthetic substances.

Phage Display can be used in conjunction with other techniques, and with enough support and studies, more applications for it can be discovered.