REPLICATION

REPLICATION: THE PROCESS:

  • Watson and Crick proposed a scheme for replication of DNA.
  • The Original statement that “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material (Watson and Crick, 1953)
  • The scheme suggested that the two strands would separate and act as template for the synthesis of new complementary strands.
  • New DNA molecule must have one parental strand and one new strand.
  • This scheme of replication is called Semiconservative type of replication.

  Experimental Proof of semiconservative nature of replication:

  • It is now proved experimentally that replication is semiconservative type.
  • It was first shown in Escherichia coli and subsequently in higher organism.
  • Mathew Messelson and Franklin Stahl performed the following experiment in 1958.

STEPS OF THE EXPERIMENTS:

  • They grew E.coli in 15NH4Cl medium for many generations. (15N is heavy nitrogen not radioactive element)
  • The result was that 15N was incorporated into newly synthesized DNA and other nitrogen containing compound as well.                                                        
  • This heavy DNA molecule could be distinguished from normal DNA by centrifugation in a cesium chloride (CsCl) density gradient.
  • Then they transferred the E.coli into a medium with normal 14NH4Cl and let them grow.(E.coli divides in 20 minutes)
  • They took samples at definite time intervals as the cells multiplied, and extracted the DNA that remained as double-stranded helices.
  • Various samples were separated independently on CsCl gradients to measure the densities of DNA.
  • The DNA that was extracted from the culture one generation after the transfer from 15N to 14N medium had a hybrid or intermediate density.
  • DNA extracted from the culture after another generation (after 40 min.) was composed of equal amount of this hybrid DNA and of ‘light ‘DNA.

Experiment by Taylor and colleagues:

  • Used radioactive thymidine to detect distribution of newly synthesized DNA in the chromosomes.
  • They performed the experiment on Vicia faba (faba beans) in 1958.
  • They proved the semiconservative nature of DNA replication in eukaryotes.

Replication Machinery and Enzymes:

  • In all living cells such as E.coli replication requires a set of enzymes.
  • E.coli completes the replication of its DNA in within 38 min.
  • The average rate of polymerization has to be approx. 2000 bp per sec.
  • The polymerization process must be accurate; any mistake during replication would result into mutation.
  • Deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, dTTP) serve dual purposes:
  • Provide energy for polymerization.
  • Acts as substrates for polymerization.
  • The replication process occurs within a small opening of the DNA helix called replication fork.
  • The region where, replication fork formed is called origin of replication.
  • The replication fork is formed by an enzyme called helicase.
  • Two separated strand is called template strands.
  • Main enzyme is DNA-dependent DNA polymerase, since it uses a DNA template to catalyze the polymerization of deoxyribonucleotides.
  • DNA polymerase catalyses polymerization only in one direction i.e. 5’→3’.
  • On one strand (template with 3’→5’ polarity) the replication is continuous hence called leading strand.
  • In another strand (template with 5’→3’ polarity) the polymerization takes place in the form of short fragment called Okazaki fragment.
  • The short fragments are joined by DNA ligase, hence called lagging strand.
  • In eukaryotes replication takes place in S-phase of cell cycle.
  • A failure of cytokinesis after replication results into polyploidy.

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