DNA Replication
Watson and Crick, Nature 1953:
'It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material'. Perhaps the most widely quoted statement from the Nature article, and not without reason!
Later, Arthur Kornberg (Washington University, St Louis) replicated DNA in-vitro, determining that offspring DNA had the same base composition as the parental type. In the now famous Meselson and Stahl (CIT) experiment the replication pattern was then shown to be semi-conservative.
In topic 2.5.1 replication of DNA in the S phase (Interphase) was described as a replication of the chromosome. In this section the mechanism for DNA replication is explained.
3.4.1 Mechanism of DNA replication
3.4.2 Conservation of the base sequence of DNA
3.4.3 Semi-conservative replication.
3.4.1 Explain DNA replication in terms of unwinding the double helix and separation of the strands by helicase,
followed by formation of the new complementary strands by DNA polymerase.(3)
Explain means to give a detailed account of causes, reasons or mechanisms.
1. The original double helix molecule.
2. Helicase enzyme breaks the hydrogen bonds between complementary base pairs. This unzips the double helix at a position called the replication fork.
3. There is an abundant supply of nucleotides in the nucleus for the formation of the new polynucleotides.
4. Nucleotides base pair to the bases in the original strands.
5. DNA polymerase joins together the nucleotides together with strong covalent phosphodiester bonds To form a new complementary polynucleotide strand.
6. The double strand reforms a double helix under the influence of an enzyme.
7 Two copies of the DNA molecule form behind the replication fork. These are the new daughter chromosomes.
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Speed of replication:
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DNA replication can take a few hours and this limits the speed of cell division.
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Bacteria can replicate quickly because of the relatively small amount of DNA.
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Eukaryotic organism's accelerate DNA replication by having thousands of replication forks along the length of the DNA molecule.
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3.4.2 Explain the significance of complementary base pairing in the conservation of the base sequence of DNA.(3)
Explain means to give a detailed account of causes, reasons or mechanisms.
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The significance of the mechanism outlined above is that the DNA molecule is copied precisely from one cell generation to the next.
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In a unicellular organism this means that the total genome is successfully copied into each new generation.
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In the multi-cellular organism all cells contain an exact copy of the total genome (even though not fully expressed).
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Genes (base sequences) are faithfully passed from one generation to the next.
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The genes (base sequences) which the reader possess have been passed from generation to generation until they arrived in you now. With minor and rare modification the base sequences copied by DNA replication and successfully passed on through sexual reproduction. Your base sequences have been copied for thousands of years.
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A worked example is provided.
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3.4.3. State that DNA replication is semiconservative.(1)
State means to give a specific name, value or other brief answer without explanation or calculation.
The mechanism of semi-conservative replication produces two descendent double helices that each contain one of the original polynucleotide chains. This need not have been the case as there are 3 possible mechanism for replication:
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Conservative replication in which the original molecule is completely retained an a new molecule (two new polynucleotide chains are combined).
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Semi-conservative replication in which each of the new double helices contains one of the original polynucleotide chains.
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Dispersive replication in which fragment of the original double helix serve as templates for the new DNA . The fragments are randomly arranged.
In 1958 Matthew Meselson and Frank Stahl ( California Institute of technology) carried out the experiment which convinced everyone that the actual mechanism was semi-conservative as originally proposed by Watson & Crick.
Experiment : They used the bacterium E. coli together with the technique of density gradient centrifugation, which separates molecules on the basis of their density.
(1) This is a calibration step in which the bacterium is cultured in with Ammonium chloride (NH4Cl )
but the nitrogen was isotope N14. Therefore both polynucleotide chains contains nucleotides which contained N14. This produced in centrifugation the 'light',( N14N14 ) chains.
(2) This is another calibration step in which the bacterium is cultured in with Ammonium chloride (NH4Cl )
but the nitrogen was isotope N15. Therefore both polynucleotide chains contains nucleotides which contained N15. This produced in centrifugation the 'heavy',( N15N15)chains.
N15N15---> N15N14
(3) The 'heavy' ( N15N15) bacteria have been returned to a medium of ammonium chloride with N14. The DNA produced was neither 'heavy' or 'light' but exactly intermediate (N15N14). The reason is that one polynucleotide is composed of bases of N14 (new one) and the other N15 (old one). This step eliminates conservative replication as a mechanism.
N15N14 ---> N14N14, N15N14
(4) The hybrid bacterium N15N14is returned to the N14medium and after 40 minutes or two generations there are 'light' and 'intermediate' DNA. This step eliminates the dispersive replication hypothesis.
The conclusion of the experiment:
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Step 3 eliminates the Conservative replication mechanism.
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Step 4 eliminates the Dispersive replication mechanism.
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In combination with step 3 with the formation of the intermediate DNA, and the elimination of conservative (step3) and dispersive (step4) replication the mechanism of semi-conservative replication is adopted.
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