DNA Replication

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  • DNA Replication
    • Why does DNA replicate?
      • DNA copies itself before cell division so that each new cell has full amount of DNA
        • semi-conservative replication
          • half of strands in each new DNA molecule are from original DNA molecule are from original DNA molecule
            • DNA copies itself before cell division so that each new cell has full amount of DNA
              • semi-conservative replication
                • half of strands in each new DNA molecule are from original DNA molecule are from original DNA molecule
                  • means theres genetic continuity between generations of cells
              • means theres genetic continuity between generations of cells
          • How is DNA replicated?
            • DNA molecule has paired base structure which makes it easy for DNA to copy itself
            • 1. enzyme DNA helicase breaks hydrogen bonds between bases on 2 polynucleotide DNA strands. Makes helix unwind to form 2 single strands
            • 2. each original strand acts as template for new strand. Complementary base pairing means that free-floating DNA nucleotides are attracted to their complementary exposed bases on each original template strand
            • 3. condensation reactions join nucleotides of new strand together - catalysed by enzyme DNA polymerase. Hydrogen bonds form between bases on original & new strands. Each new DNA molecule contains 1 strand from original DNA molecule & 1 new strand
          • Action of DNA polymerase
            • each end of DNA strand is slightly different in structure
              • one end is called 3 prime end & one end is called 5 prime end
                • active site of DNA polymerase only complementary to 3 prime end of newly forming DNA strand
                  • enzyme can only add nucleotides to new strand at 3 prime end
                    • new strand made in 5 prime to 3 prime direction & DNA polymerase moves down template strand in 3 prime to 5 prime direction
                      • because strands are antiparallel DNA polymerase moves in opposite direction to DNA polymerase working on other template strand
          • Evidence for semi-conservative replication
            • Watson & Crick came up with theory
              • wasn't until Meselson & Stahl's experiment that it was validated
                • before - people were unsure if replication was semi-conservative or conservative
                • showed DNA replicated using semi conservative method
                • used 2 isotopes of nitrogen (DNA contains nitrogen) - heavy 15N & light 14N
                • 1. 2 samples of bacteria grown for many generations - 1 in nutrient broth containing light N & 1 in broth with heavy N. As bacteria reproduced - took up nitrogen from broth to help make nucleotides for new DNA. So N gradually became part of bacteria's DNA
                • 2. sample of DNA taken from each batch & spun in centrifuge. DNA from heavy N bacteria settled lower down centrifuge tube than DNA from light N bacteria - it is heavier
                • 3. bacteria grown in heavy N broth taken out & put in broth containing only light N. Bacteria left for 1 round of DNA replication & another DNA sample was taken & spun in centrifuge
                • 4. if replication was conservative the original heavy DNA which would still be together would settle at bottom & new .light DNA would settle at top
                • 5. if replication was semi-conservative - new bacteria molecules would contain 1 strand of old heavy DNA & 1 new strand of light DNA. So new DNA would  settle out between where light N DNA settled out & where heave N DNA settled out
                • 6. DNA settled in middle showing that DNA molecules contained mixture of heavy & light N. The bacterial DNA replicated semi-conservatively in light N

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