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Replication of DNA

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Replication of DNA is the process of formation of carbon copies of a DNA. DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. For this, DNA functions as its own template. Therefore, DNA replication is an autocatalytic function of DNA. It usually occur during S-phase of the cell cycle.

After proposing the double helical structure for DNA, Watson and Crick had immediately proposed a scheme for replication of DNA, which is known as semiconservative DNA replication.

SEMICONSERVATIVE DNA REPLICATION


The scheme suggests that the two strands of DNA would separate. Each separated serves as a template (model or guide) for the formation of a new but complementary strand. Thus, the new or daughter DNA molecules formed would be made of one old or parental strand and another newly formed complementary strand. This method of formation of new daughter DNA molecules is called semi-conservative method of replication.

EXPERIMENTAL PROOF

 

The following experiment suggests that DNA replication is semi-conservative.

 

1. Matthew Meselson and Franklin Stahl (1958) conducted experiments using heavy nitrogen (N15) to determine whether the concept of semi-conservative replication is correct. They used Cesium chloride (CsCl2) gradient centrifugation technique for this purpose. A dense solution of CsCl2, on centrifugation, forms density gradient-bands of a solution of lower density at the top that increases gradually towards bottom with highest density. If DNAs of different densities are mixed with CsCl2 solution, these would separate from one another and would form a definite density band in the gradient along with CsCl2solution.

 

Meselson and Stahl created DNA molecules of different densities by using normal nitrogen N14 and its heavy isotope N15. For this purpose, Escherichia coli was grown in NH4C1 containing culture medium for many generations, so that bacterial DNA become completely heavy. This radioactive or heavy DNA (incorporating N15) had more density than DNA with normal nitrogen (N14). The bacteria were then transferred to culture medium containing only normal nitrogen (N14H4CI). The change in density was observed by taking DNA samples periodically.

If DNA replicates semi-conservatively, then each heavy (N15) DNA strands should separate and each separated strand should acquire a light (N14) partner after one round of replication. This should be a hybrid DNA made of two strands, i.e., N14-N15. Meselson and Stahl observed that such DNA was actually half dense indicating the presence of hybrid DNA molecules. After second round of replication there would be four DNA molecules. Of these, two molecules would be hybrid (N14-N15) showing half density as earlier and the remaining two molecules would be made of light strands (N14-N14) Thus, after second generation, the same half dense band (N14-N15) was seen but the density of light bands (N14-N14) increased. Meselson and Stahl's work as such provided confirmation of Watson-Crick model of DNA and its semi-conservative replication.

 

2.Autoradiography experiment in Vicia faba.:-Autoradiography was also utilized by J.H. Taylor and his co-workers for the study of duplicating chromosomes in the root tip cells of Vicia faba. Results of such experiments were first published in 1957. After incorporation of tritiated thymidine, when root tips were transferred to unlabeled medium, in the first generation of duplication both chromatid were labeled (interpreted as one DNA double helix in each chromatid, and only one of the two strands labeled). In the second cycle of replication, in each chromosome, one of the two chromatid was found to be labeled. This was interpreted as showing semi-conservative mode of replication.

 

ENZYMES INVOLVED IN DNA REPLICATION

 

The process of DNA replication require a set of catalysts (enzymes). The main enzyme for DNA replication is DNA dependent DNA polymerase. The other enzymes required for replication are primase, helicase, single strand binding protein, DNA ligase, topoisomerase, etc. A few are described below.

 

1. DNA polymerases :-

These enzymes copy DNA sequences by using one strand as a template. The reaction catalyzed by DNA polymerases is the addition of deoxyribonucleotides to a DNA chain by using dNTPs as substrates.

All DNA polymerases require a template strand, which is copied (that is why it is also known as DNA dependent DNA polymerase). DNA polymerases also require a primer, which is complementary to the template. The reaction of DNA polymerases is thus better understood as the addition of nucleotides to a primer to make a sequence complementary to a template. The requirement for template and primer are exactly what would be expected of a replication enzyme. Because DNA is the information store of the cell, any ability of DNA polymerases to make DNA sequences from nothing would lead to the degradation of the cell's information copy.

More than one DNA polymerase exists in each cell. The key distinction among the enzyme forms is their processitivity—how long a chain they synthesize before falling off the template. A DNA polymerase used in replication is more processive than a repair enzyme. The replication enzyme needs to make a long enough chain to replicate the entire chromosome. The repair enzyme needs only to make a long enough strand to replace the damaged sequences in the chromosome. The best-studied bacterium, E. coli, has three DNA polymerase types. :-

 

DNA polymerase I (Pol I) is primarily a repair enzyme, although it also has a function in replication. About 400 Pol I molecules exist in a single bacterium. DNA polymerase I only makes an average of 20 phosphodiester bonds before dissociating from the template. These properties make good sense for an enzyme that is going to replace damaged DNA. Damage occurs at separate locations so the large number of Pol I molecules means that a repair enzyme is always close at hand.

 

DNA polymerase II is a specialized repair enzyme. Like Pol I, a large number of Pol II molecules reside in the cell (about 100). The enzyme is more processive than Pol I. Pol II has the same editing (3′ to 5′) activity as Pol I, but not the 5′ to 3′ exonuclease activity.

 

DNA polymerase III :-The actual replication enzyme in E. coli is DNA polymerase III. Its properties contrast with Pol I and Pol II in several respects. Pol III is much more processive than the other enzymes, making about 500,000 phosphodiester bonds on the average. In other words, it is about 5,000 times more processive than Pol I and 50 times more processive than Pol II. Pol III is a multi subunit enzyme. It lacks a 5′ to 3′ exonucleolytic activity, although a subunit of the enzyme carries out the editing (3′ to 5′) function during replication. Finally, only about 10 molecules of Pol III reside in each cell. This remains consistent with the function of Pol III in replication, because the chromosome only needs to be copied once per generation. Therefore, the cell only requires a few molecules of the enzyme. Pol III synthesizes DNA at least a hundred times more rapidly than the other polymerases. It can synthesize half of the bacterial chromosome in a little more than 20 minutes, which is the fastest that the bacterium can replicate.

 

2. Primase :- Primase is an enzyme that synthesizes short RNA sequences called primers. These primers serve as a starting point for DNA synthesis. Since primase produces RNA molecules, the enzyme is a type of RNA polymerase. Primase functions by synthesizing short RNA sequences that are complementary to a single-stranded piece of DNA, which serves as its template. It is critical that primers are synthesized by primase before DNA replication can occur. This is because the enzymes that synthesize DNA, which are called DNA polymerases, can only attach new DNA nucleotides to an existing strand of nucleotides. Therefore, primase serves to prime and lay a foundation for DNA synthesis.

 

3. Helicase :- Helicases are enzymes that bind and may even remodel nucleic acid or nucleic acid protein complexes. There are DNA and RNA helicases. DNA helicases are essential during DNA replication because they separate double-stranded DNA into single strands allowing each strand to be copied. During DNA replication, DNA helicases unwind DNA at positions called origins where synthesis will be initiated. DNA helicase continues to unwind the DNA forming a structure called the replication fork, which is named for the forked appearance of the two strands of DNA as they are un-zipped apart. The process of breaking the hydrogen bonds between the nucleotide base pairs in double-stranded DNA requires energy. To break the bonds, helicases use the energy stored in a molecule called ATP, which serves as the energy currency of cells. DNA helicases also function in other cellular processes where double-stranded DNA must be separated, including DNA repair and transcription.

 

4. Single Strand Binding Protein :- Single-stranded DNA-binding protein, or SSB, binds to single-stranded regions of DNA to prevent premature annealing, to protect the single-stranded DNA from being digested by nucleases, and to remove secondary structure from the DNA to allow other enzymes to function effectively upon it. Single-stranded DNA is produced during all aspects of DNA metabolism: replication, recombination and repair. As well as stabilizing this single-stranded DNA, SSB proteins bind to and modulate the function of numerous proteins involved in all of these processes.

5. DNA Ligase :- DNA ligase is a specific type of enzyme, that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It helps in maturation of lagging strand by joining Okazaki fragments.

 

6. Topoisomerase :- are enzymes that regulate the over winding or under winding of DNA. The winding problem of DNA arises due to the intertwined nature of its double helical structure. For example, during DNA replication, DNA becomes over wound ahead of a replication fork. If left unabated, this tension would eventually grind replication to a halt .

In order to help overcome these types of topological problems caused by the double helix, topoisomerases bind to either single-stranded or double-stranded DNA and cut the phosphate backbone of the DNA. This intermediate break allows the DNA to be untangled or unwound, and, at the end of these processes, the DNA backbone is resealed again. Since the overall chemical composition and connectivity of the DNA does not change, the tangled and untangled DNAs are chemical isomers, differing only in their global topology, thus their name. Topoisomerases are isomerase enzymes that act on the topology of DNA.

MECHANISIM OF DNA REPLICATION

 

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

 
ORIGIN OF REPLICATION

 
DNA Replication process is initiated at particular points in the DNA, known as "origins", which are targeted by proteins that separate the two strands and initiate DNA synthesis. Origins contain DNA sequences recognized by replication initiator proteins. These initiators recruit other proteins to separate the strands and initiate replication forks.Replication of DNA in E. coli always begins at a definite site called origin of replication. On the other hand, eukaryotes have several thousand origins of replication.

 

 

1. Initiation :- DNA replication requires that a double helical parental molecule is unwound so that its internal bases are available to the replication enzymes. Unwinding is brought about by enzyme `helicase', which is ATP dependent.

Unwinding of DNA molecule into two strands results in the formation of Y-shaped structure, called replication fork. These exposed single strands are stabilized by a protein known as single-strand binding protein (SSB). Due to unwinding a super coiling get developed on the end of DNA opposite to replicating fork. This tension is released by enzyme topoisomerase.

Once the DNA becomes single-stranded, single-strand binding protein (SSBP) binds the DNA, which prevents the two strands from re-annealing. To a new strand is II now to be synthesized opposite to the parental strands. DNA polymerase is the true replicase , which is incapable of initiating DNA synthesis, i.e., it is unable to deposit the first nucleotide in a daughter (new) strand. Another enzyme, known as primase, synthesizes a short primer strand of RNA. The primer strand then serves as a stepping stone (to start errorless replication). Once the initiation of DNA synthesis is completed, this primer RNA strand is then removed enzymatically.

2. Elongation :- Once the primer strand is formed, DNA replication occurs in 5' -> 3' direction, i.e., during synthesis of a new strand, deoxyribonucleotides (dATP, dGTP, dTTP, dCTP) are added only to the free 3'OH end. Thus, the nucleotide at 3' end is always the most recently added nucleotide to the chain.

Because only one strand can serve as a template for synthesis in the 5′ to 3′ direction (the template goes in the 3′ to 5′ direction, because the double helix is antiparallel), only one strand, the leading strand, can be elongated continuously. Ahead of the replication fork, topoisomerase helps unwind the DNA double helix and keep the double strands from tangling during replication.

Synthesis of the second (lagging) strand is more complicated because it is going in the wrong direction to serve as a template. No DNA polymerase exists to synthesize DNA in the 3′ to 5′ direction, so copying of the lagging strand is discontinuous—that is, short strands of DNA are made and subsequently matured by joining them together. An RNA primer, which is made by primase, initiates each of these small pieces of DNA. Then DNA polymerase III elongates the primer until it butts up against the 5′ end of the next primer molecule. This is called lagging daughter strand. These short pieces of DNA are known as Okazaki fragments, these segments are about 1,000 -2,000 nucleotides long in prokaryotes. Hence DNA replication is semi-discontinuous. DNA polymerase I then uses its polymerizing and 5′ to 3′ exonuclease activities to remove the RNA primer and fill in this sequence with DNA. Because Pol I is not very processive, it falls off the lagging strand after a relatively short-length synthesis. DNA polymerases can't seal up the nicks that result from the replacement of RNA primers with DNA. Instead, another enzyme, DNA ligase, seals off the nicks by using high energy phosphodiester bonds in ATP or NAD to join a free 3′ hydroxyl with an adjacent 5′ phosphate.

3. Termination :-

Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome; these are not known to be regulated in any particular way.

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.






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Last Updated on Monday, 26 November 2012 22:22

Word of Wisdom

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A discipline is simply not needed where there is love. -         Sri Sri Ravishankar


 

“After just 5 weeks of daily 5-to-16-minute training sessions in meditation, Research subjects showed strong brain-wave changes associated with positive emotions” - Harvard Business Review


For more info, www.facebook.com/meditatingmonday

 

 

 

 

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Adrenal Gland

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The adrenals are two glands, each sitting like a cap on top of a kidney. The adrenals are divided into two distinct regions: the cortex (outer layer) and the medulla (inner layer). The cortex makes up about 80 percent of each adrenal. The adrenals help the body adapt to stressful situations.

The cortex secretes about 30 steroid hormones. The most important of these are cortisol and aldosterone. Cortisal regulates the body's metabolism of carbohydrates, proteins, and fats. Aldosterone regulates the body's water and salt balance. The cortex is extremely important to bodily processes. If it stops functioning, death occurs in just a few days.

The medulla secretes the hormones adrenaline and nor adrenaline. Both of these hormones are released during dangerous or stressful situations. They increase heart rate, blood pressure, blood flow to the muscles, blood sugar levels, and other processes that prepare a body for vigorous action, such as in an emergency.

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Last Updated on Friday, 02 December 2011 17:28

Hypothalamus-Endocrine System

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Region of the brain lying below the thalamus and making up the floor of the third cerebral ventricle. The hypothalamus contains a control centre for many functions of the autonomic nervous system, and it has effects on the endocrine system because of its complex interaction with the pituitary gland, which lies beneath it.

The hypothalamus and pituitary gland are connected by both nervous and chemical pathways. Nerve tracts from the hypothalamus stimulate the release of oxytosin and vasopressin from the posterior pituitary gland; these hormones cause smooth-muscle contractions in the circulatory and reproductive systems. Hormones secreted from the hypothalamus trigger the release from the anterior pituitary of growth hormone, adrenocorticotropic hormone, luteinizing hormone, and other pituitary secretions.

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Last Updated on Friday, 02 December 2011 17:39

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