Topic 11, Replication and Recombination of DNA

  1. Introduction, quote from, Watson and Crick's 1953 paper (on handout)

  2. Semiconservative, conservative, and dispersive models of DNA replication

  3. Chromosome (DNA) replication in vivo in a higher plant (Vicia faba)
    (Taylor, Woods, and Hughes, 1957, described on handout and in book)
    A. Used tritiated thymidine (3H) which is found in DNA and no other molecule
    B. Grew cells for 1 cell cycle in the presence of tritiated thymidine and
         autoradiographed the cells
        1. Found that both chromatids of each chromosome were labeled
    C. Then grew cells for a second generation in medium that did not contain
          tritiated thymidine
        1. Found that only one chromatid of each chromosome was labeled
    D. These observations indicated that the DNA in the chromosomes replicated by
         semi-conservative replication (see handout and text)

  4. Meselson and Stahl, 1968, replication of DNA in E. coli
    A. 14N is the normal isotope of nitrogen and 15N is a heavy isotope (contains one
         additional neutron)
    B. Grew E. coli in medium where 14N was the only nitrogen source or medium
         where 15N was the only nitrogen source
    C. Extracted DNAs from 14N and 15N grown bacteria, placed the DNAs in a CsCl solution,
         and spun the DNA to equilibrium in a centrifuge (sedimentation equilibrium centrifugation)
    D. Photographed the spinning tubes with UV 260 nm light to determine the position of the DNA in the tube
    E. Found that the 14N DNA and 15N DNA came to equilibrium at different positions in the centrifuge tubes
    F. Then took bacteria that had been grown for several generations in 15N and placed them in 14N medium
    G. After one DNA replication in 14N medium, there was a single band at an intermediate position
    H. After two DNA replications in 14N medium, there were two bands, one at the 14N position and one at the
         intermediate position
    I. These results are the results expected if DNA replicates by semi-conservative replication
    1.
  5. Origins, forks, and units of replication
    A. Definitions
        1. Origin of replication = site on a chromosome where DNA replication begins
        2. Replication fork = point at which DNA is being replicated
        3. Unit of replication (replicon) = length of DNA that is replicated following one
             initiation event
    B. DNA replication in bacteria, discussed in lecture and in text
        1. DNA replication begins at one origin of replication and proceeds in both
            directions (2 replication forks) along the entire length of the bacterial
            chromosome until the complete bacterial chromosome is replicated
    C. DNA polymerase
         1. Discovered by Kornberg et al., 1957, an enzyme from E. coli
         2. An enzyme that could catalyze DNA replication in vitro (outside of a living cell)
         3. If DNA polymerase, all 4 deoxyribonucleoside triphosphates, and template DNA
             were mixed together, DNA replication occurred
         4. If any one of these was left out, DNA replication did not occur
         5. Chain elongation was in the 5' --> 3' direction by adding one nucleotide at a time
              to the growing 3' end
         6. Since that time, two additional DNA polymerases have been discovered in E. coli,
              and DNA polymerase III is thought to be primarily involved in DNA replication
    D. Okazaki fragments (1968)
         1. The two DNA strands have opposite polarity,
              a. The 5' --> 3' strand (leading strand) is synthesized continuously
              b. The 3' --> 5' strand (lagging strand) is synthesized discontinuously
                   as Okazaki fragments
         2. A RNA primer molecule is added to the beginning of each DNA fragment
             being synthesized by RNA primase
         3. The Okazaki fragments are joined together by DNA ligase
    E. Proofreading and error correction occurs during synthesis to correct mistakes

  6. DNA replication in eukaryotes
    A. Eukaryotes contain far more DNA per cell than prokaryotes and DNA synthesis is
         much slower at each replication fork
    B. There are very large numbers of replicons per genome of eukaryotic cells
         (~25,000 per mammalian genome)
    C. In eukaryotes, the Okazaki fragments are much smaller (about 10 x as small) than
         in prokaryotes

  7. Telomeres, the differentiated ends of chromosomes
    A. A repeated sequence of bases is present at the ends of each eukaryotic
         chromosome
         1. In ciliated protozoa, the sequence 5'TTGGGG3' is repeated hundreds of times
    B. An enzyme, telomerase, replicates the telomeric sequences
    C. There is considerable evidence that the number of telomeric sequences
          becomes smaller as cells divide
    D. In cancer cells, the number of telomeric sequences is not reduced as cells divide

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        Updated 11/13/00