Wednesday 24 April 2019

DNA replication

                                                          REPLICATION OF DNA

YOUTUBE video  link of DNA replication from  MAKE IT SIMPLE ;-

INTRODUCTION - DNA can self replicates. DNA replication is biological process of producing two identicak replicas of DNA from one origenal DNA molecules. process occurs in living organism biological inheritance . 

 In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule.  The cell possesses the distinctive property of division, which makes replication of DNA essential.
DNA is made up of a double helix of two complementary strands. During replication, these strands are separated. Each strand of the original DNA molecule then serves as a template for the production of its counterpart, a proce.9+ss referred to as semiconservative replication. As a result of semi-conservative -\replication, the new helix will be composed of an original DNA strand as well as a newly synthesized strand. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.In a cell, DNA replication begins at specific locations, or origins of replication, in the genome.
Unwinding of DNA at the origin and synthesis of new strands, accommodated by an enzyme known as helicase, results in replication forks growing bi-directionally from the origin. A number of proteins are associated with the replication fork to help in the initiation and continuation of DNA synthesis. Most prominently, DNA polymerasesynthesizes the new strands by adding nucleotides that complement each (template) strand. DNA replication occurs during the S-stage of interphase.
DNA replication (DNA amplification) can also be performed in vitro (artificially, outside a cell). DNA polymerases isolated from cells and artificial DNA primers can be used to start DNA synthesis at known sequences in a template DNA molecule. 

DNA polymerase

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

Replication process

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


For a cell to divide, it must first replicate its DNA.This process is initiated at particular points in the DNA, known as "origins", which are targeted by initiator proteins. In E. coli this protein is DnaA; in yeast, this is the origin recognition complex. 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. Once the origin has been located, these initiators recruit other proteins and form the pre-replication complex, which unwinds the double-stranded DNA.


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 DnaGprotein superfamily which contains a catalytic domain of the TOPRIM fold type. 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.
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. In eukaryotes, leading strand synthesis is thought to be conducted by Pol ε; however, this view has recently been challenged, suggesting a role for PolPrimer removal is completed Pol  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.

Diagram, Dna, Biology, Labeled, Replication, Label

Replication fork

Many enzymes are involved in the DNA replication fork.
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 always 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 nascent (new) lagging strand DNA, whose direction of synthesis is opposite to the direction of the growing replication fork.

Leading strand

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

Lagging strand

The lagging strand is the strand of nascent 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

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. 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.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.

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.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.
Sequence of enzyme involved in DNA REPLICATION.... Here is your answer
 1st - primase
 2nd - helicase (splits the DNA)
 3rd - DNA polymerase (synthesis new strand)
4th - ssb protein (maintaining the sepretes DNA strand)
5th - topoisomarase (open the knot or overwinding or supercoiling during dna replication)
6th - DNA ligase(joining of the dna fragments). 

Replication machinery

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.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. 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 nascent 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.
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.


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.

Sunday 21 April 2019

DNA: Notes on DNA | Nucleic Acid

The below mentioned article provides a note on DNA:- 1. DNA Chemistry 2. DNA Replication 3. Carrier of Genetic Informa­tion.

DNA Chemistry:

DNA is found to consist of thread-like particles of high molecular weight in isolated condition.
A number of units called nucleotides become linked together and form the nucleic acid. A nucleotide is composed of a molecule of Pentose (5-carbon sugar), a nitrogen base (Purine and Pyrimidine) and phosphoric acid.
The purine bases are adenine and guanine and the pyrimidine bases are cytosine and thymine. The diff­erence in the chemical composition of DNA is due mainly to the difference in arrangement of the nitrogen bases.
In an intact DNA molecule, base remains attached to a 5-carbon sugar (Deoxyribose) to form a deoxynucleoside. On analysis of DNA samples it has been found that the bases do not occur at a random fashion and that the sum of the purines is equal to the sum of the pyrimidines. Further, it is seen that the content of adenine equals the content of thymine and the content of guanine equals the con­tent of cytosine.
Model of Watson and Crick:
Watson and Crick (1953) have construct­ed a most satisfying three dimensional model of DNA. According to this model, a DNA molecule consists of two long chains.
The chains run in opposite direction and are coiled round each other forming a double helix (Fig. 2.7). Each chain consists of a nitrogen base attached to pentose and the pentose in its turn is attached to the phosphoric acid. The bases of one chain are joined with those of the other chain by hydrogen bond. But the bondage bet­ween the bases is not at random and their sequences form a regular pattern.
Adenine is always linked with Thymine and Cytosine is always linked with Guanine. Thus the sequence of the bases of one strand determines the sequence of the bases of the other strand and this arrangement makes the strands complementary.
Helical Structure of Chromosome
It is believed that the base pairs carry all the genetic information in an, organism. Thus the four alphabets A, T, C and G (A=adenine, T=thymine, C=cytosine, G=guanine) carry all the codes for the genetical specifications of all organisms. However, it is not known how many base pairs make up a gene.

 DNA Replication:

In 1950, Chargaff showed that in any DNA, the amount of adenine equals to the amount of thymine and the amount of gua­nine equals to the amount of cytosine. Using X-ray crystallography technique, Watson and Crick in 1953 suggested that the DNA molecule consists of two polynucleotide strands inter-wined to form a double helix. The two strands are linked together by weak hydrogen bonds bet­ween the paired bases.
They further ad­vocated that:
(1) the two polynucleotide strands run in opposite directions to give the observed symmetry and
(2) that pair­ing takes place between one Purine base and one Pyrimidine base.
Otherwise the links would become either too small or too large. This theory has well been substantiated from studies from X-ray diffraction, elec­tron micrograph and enzymatic studies. It is now accepted as a working hypothesis.
The strong point in favour of the hy­pothesis is that it offers an explanation of how replication is affected when the chromosomes appear as double structures in the prophase stage of mitosis and meiosis.
To quote Watson and Crick:
“Our model of deoxyribose nucleic acid is, in effect a pair of templates, each of which is complementary to the other. We imagine that prior to duplication the hydrogen bonds are broken, and the two chains un­wind and separate. Each chain then-acts as a template for the formation on itself a new companion chain, so that eventually we shall have two pairs of chains where we had only one before. Moreover, the sequ­ences of the pairs of bases will have been duplicated exactly” (Figs. 2.8 and 2.9).
Unwind and Duplication of DNA
The experimental support to it:

A good experimental support for the correctness of the explanation came from the work of Meselson and Staht in 1958 on Escherichia coli. The bacterium when grown in a medium with a nitrogen source containing only 15N, this heavy isotope’ becomes incorporated in the new DNA formed.
This can be located because the DNA is now denser than 14N and it can be separated by centrifugation and its sedim­entation rate measured.
The bacteria are then transferred to a medium containing only14N and the DNA is analysed at each successive doubling of the chromo­somes. In such analysis three kinds of DNA were recognised—15N DNA, 14N DNA and an intermediate form 15N/14N DNA (Fig. 2.10). The result thus shows complete agreement to the model of Watson and Crick.
Synthesis of DNA

DNA as Carrier of Genetic Informa­tion:

The chromosomes consist of proteins, DNA and RNA. In the search for likely genetic material, the proteins were first of all regarded as likely candidates. However, in 1944 work on Diplococcus pneumoniae, a bacterium causing pneumonia, was carried out in America by Avery and his collaborators and their work showed that DNA must carry the genetic infor­mation.
Since then a good number of evidences have emerged out to prove that DNA is the carrier of genetic information.
They are discussed below:

Transformation of bacteria:
Certain strains of Diplococcus pneumoniae are avirulent and if injected into an animal would not cause the disease. Similarly if a virulent strain is first killed by heat treatment and then injected it would not produce the disease.
But if the animal is inoculated with a mixture of these two types (Avirulent and heat killed), the animal shows symptoms of the disease and the avirulent type has now been transformed into viru­lent type.
This genetic change is known as ‘Transformation’ when carried out in test-tube condition. The other known evidence in favour of DNA’s role as heredi­tary determinant in metabolism comes from the studies of the transformation of Pneumococci bacteria. Experiments have estab­lished that DNA from one strain of this bacteria can alter the inherited metabolic properties of a second strain of bacteria.
By the application of mutation technique a strain of Pneumococcus can be produced which is incapable of utilising Mannitol. These bacteria lack in the enzyme, Mannitol-phosphate-dehydrogenase and are de­signated as M cells.
If DNA is isolated from M+ cells and is introduced in a culture of Mcells, many M cells become trans­formed into M+ cells and the progeny of the transformed cells becomes capable of producing the enzyme.
The result proves in a convincing way that the hereditary capacity of the cells can be permanently altered. Further, the experiment makes it clear that at least a part of the genetical material in DNA is taken up by the chromosomes of the host and in certain percentage of the cases this incorporated material is replicated in subsequent generation.
However, the me­chanism by which the DNA is incorporat­ed into a permanent functional structure is not known.
Certain strains of bacteria are resistant to antibiotics such as Penicillin. If purified DNA, extracted from a Penicillin resistant strain of Pneumococcus is added to a culture medium in which Penicillin sensitive Pneumococcus are being cultured, trans­formation takes place and the whole cul­ture becomes Penicillin resistant.
Transformation has also been studied in the bacterium Bacillus subtilis. A strain of this bacterium is unable to synthesize Try­ptophan and as such cannot thrive in a culture medium lacking the amino acid Tryptophan. If such a culture is treated with DNA from a normal strain, a num­ber of ‘Transformants’ appear in the Tryptophan-free medium. From these, many colonies of the new strain arise since the trait is heritable.
Many such experiments involving the transformation of genetic character have been made. These experiments have prov­ed that the transforming agent is DNA and that DNA is the major chemical that controls the synthesis and operation of enzymatic machinery from generation to generation.
Bacteriophage infection:
Identification of DNA as the genetic messenger is also proved by experiments with virus. Bac­teriophage is a virus on bacteria and is popularly called phage. The phage is made up of two parts —a head and a tail. The DNA of the phage lies in the head.
Both the head and tail remain capsulated in a pro­tein coat. During infection the phage be­comes attached to the bacterial cell wall and injects its DNA into the bacteria. Inside the bacterial cell, the DNA of the phage takes over the metabolic machinery and starts producing phage DNA and phage protein like its own kind.
Finally, the protein combines with the DNA to form complete phages. After some time the bacteria lyse and break releasing a large number of new phages.
In an experiment the DNA of the phage was lebelled with 35P and these were used to infect the bacteria. The result showed that the host DNA became radio-active. This proves that the DNA of the phage enters the DNA of the bacteria forming new phages at the expense of the host bac­teria. The life cycle of the phage is present­ed in Fig. 2.11.
Life Cycle of Bacteriophage
The evidence proves beyond doubt that a foreign DNA is capable of influencing the synthetic capacities of the host cell. This has led us to believe that an altered DNA or virus DNA is responsible for many diseases specially cancer.
Transduction is an ex­perimental device by which genetic charac­teristics of one bacterium is transferred to another bacterium by using a third party. This third party in transduction is a phage. Thus when a bacterium sensitive to Strepto­mycin is injected with phages grown pre­viously on bacterial cells resistant to Streptomycin, a small portion of the sensi­tive cells becomes Streptomycin resistant.
The resistant cells here are donors and sensitive cells are recipients and the third party is phage. While multiplying, in the donor cell the phage incorporated itself with some of the donor’s DNA which is Streptomycin resistant. The phage when transduced, this resistant and incorporat­ed portion of the host DNA into the reci­pients DNA made some of the recipients DNA Streptomycin resistant.


  • Biotechnology deals with the techniques of using living organisms or enzymes from organisms to produce products useful to humans.
  • The processes like in vitro fertilization leading to a ‘test-tube’ baby, synthesizing a gene and using it, developing a DNA vaccine or correcting a defective gene, are all parts of biotechnology.
  • Biotechnology can be defined as- ‘The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services’.
Fig. test tube baby