Sunday, 27 September 2020

Semiconservative Replication and Meselson and Stahl Experiment

Semiconservative Replication and Meselson and Stahl Experiment

This model of DNA replication was proposed by Watson and Crick. According to this model of DNA replication, both strands of parental DNA separate from each other. Each old strand synthesise  synthesizes a new strand. Thus each of the two resulting DNA molecules has one parental and one new strand. This model of DNA replication universally accepted because there are several evidences in support of this mode.


The semi-conservative model (mechanism) of DNA replication consists of six important steps, viz :
• Unwinding,
• Binding of RNA primase, 
• Elongation,
• Removal of primers,
• Termination, and
• DNA repair.

These are briefly discussed as follows


* Unwinding :
The first major step in the process of DNA, replication is the breaking of hydrogen bonds between bases of the two anti-parallel strands. The unwinding of the two strands is the starting point The splitting happens in places of the chains which are rich in A- T.
  That is because there are only two bonds between Adeninie and Thymine, whereas there are three hydrogen bonds between Cytosine and Guanine. The Helicase enzyme splits the two strands. The initiation paint where the splitting starta is called "origin of replication". The stracture that is created is known as "Replication Fork".


* Binding of RNA Primase : 
Synthesis of RNA primer is essential for initiation of DNA replication. RNA primer in synthesized by DNA template near the origin with the help of RNA Primase. RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3'➡5' strand due to the hydrogen bonds between the bases. RNA nucleotides are the primers (starters) for the binding of DNA nucleotides.


* Elongation :
The elongation proceeds in both directions, viz. 5'➡3' and 3'➡5' template. The 3'➡5' proceeding daughter strand that uses a 5'➡3' template is called leading strand because DNA Polymerase 'a' can "read" the template and continuously add nucleotides. The 3'➡5' template cannot be "read" by DNA Polymerase 'a'. The replication of this template is complicated and the new strand is called lagging strand.
  In the lagging strand the RNA Primase adds more RNA Primers. DNA polymerase a reads the template and lengthens the bubbles The gap between two RNA primer is called "Okazaki" Fragmenta. The RNA Primers are necessary for DNA Polymerase a to bind Nucleotides to the 3' end of them. The daughter strand is elongated with the binding of more DNA nucleotide.


* Removal of Primers :
The RNA Primers are removed or degraded hy DNA polymerase-I This enzyme also catalyzes the synthesis of short DNA segments to replace the primers. The gaps are filled with the action of DNA Polymerase which adds complementary nucleotides to the gaps.
The DNA Lygase enzyme adds phosphate in the remaining gaps of the phosphate-sugar backhone Each new double helix is consisted of one old and one new chain. This is called semi-conservative replication.


* Termination:
The termination takes place when the DNA Polymerase reaches to an end of the strands. In other words, it is the separation of replicated linear DNA. After removal of the RNA primer, it is not possible for the DNA Polymerase to seal the gap because there is no primer.   

Hence, the end of the parental strand where the last primer binds is not replicated. These ends of linear (chromosomal) DNA consist of noncoding DNA that contains repeat sequences and are called telomeres A part of the telomere is removed in every cycle of DNA Replication.


* DNA Repair :
The DNA replication is not completed without DNA repair. The possible errors caused during the DNA replication are repaired by DNA repair mechanism. Enzymes like nucleases remove the wrong nucleotides and the DNA Polymerase filla the gaps, Similar processes also happen during the steps of DNA Replication of prokaryotes though there are some differences.



Meselson and Stahl Experiment [1958] :

Meselson and Stahl conducted their experiment with common bacteria of human intestine i.e. Escherichia coli.

They raised E. coli on ammonium chloride having heavy isotope of nitrogen (N15) for several generations so that both the strands of DNA became labelled with N15. The bacteria were then grown in culture medium having ammonium chloride with normal nitrogen (N14). The bacteria were tested for heavy nitrogen N15 in every successive generation through density gradient centrifugation using cesium chloride. DNA of the first generation was intermediate between N15 and N14 while in second generation 50% bacteria had N14 and remaining 50% intermediate. This is possible only if DNA strands do not break up but separate for multiplication with one old strand producing a new complementary strand.