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					DNA Structure and
  Replication
    DNA is the Genetic Material
• DNA and RNA were first described by Friedrich Miescher
  in 1869. He isolated a phosphorus-containing material
  from the nuclei of cells found in pus from discarded
  surgical bandages, and called in “nuclein”. He later
  found the same material in salmon sperm. Later it was
  recognized that DNA and RNA are slightly different in
  structure.
• In pre-digital days it seemed impossible for complex
  phenotypic traits to be coded in a simple linear fashion.
  DNA was known to be a linear polymer of just
  4nucleotides, and it was thought to be just a scaffold for
  the actual genes. Proteins were considered the most
  likely genetic material, or perhaps some undiscovered
  substance.
• Definitive proof of DNA’s central role came in the 1950’s,
  but important experiments were done earlier.
     Transformation Experiments
•   In 1928, F. Griffith did a series of experiments on mice infected with
    Streptococcus bacteria. These experiments were followed up Avery,
    MacLeod, and McCarty in 1943, who demonstrated that at least in this case,
    DNA was the hereditary material, and not proteins.
•   Griffith had 2 strains of Streptococcus. The S strain had a polysaccharide
    coat around each cell, causing the colonies to have a smooth, glossy
    appearance. The R strain lacked the coat, and its colonies had a rough
    appearance. More importantly, the S strain was virulent: when injected into
    mice, they developed pneumonia and died. The R strain was avirulent: it
    did not kill the mice upon injection.
•   When S cells were killed by heat, injecting them had no effect on the mice.
    Heat killed R cells also had no effect.
•   The surprising result: when live R cells were mixed with heat-killed S cells
    and injected, the mice developed an infection and died. When bacteria
    were isolated from the dead mice, they were found to be S type.
•   Conclusion: something from the dead S cells had “transformed” the live R
    cells into S.
         More Transformation
• Avery, MacLeod, and MacCarty carried this
  result further by fractionating the dead S cells.
  They mixed various components (such as lipids,
  polysaccharides, protein, nucleic acids) of the S
  cells with live R cells, to determine which
  component caused the transformation. They
  found the DNA by itself caused the
  transformation, and no other component had
  any effect.
• This demonstrated that DNA was the hereditary
  material, but their results were not considered to
  be generally applicable to inheritance.
      Hershey-Chase Experiment
•   Another experiment, performed by Hershey and Chase in 1952,
    demonstrated the essential role of DNA using bacteriophage. At the time, a
    group of physicists who had previously worked on nuclear energy were
    moving into biology. They started work on the genetics of bacteria and
    bacteriophage that grew into modern molecular biology.
•   An important element of the Hershey-Chase experiment is that DNA
    contains phosphorus but not sulfur, while protein contains sulfur but not
    phosphorus. Thus it is possible to label the two types of molecule
    independently, with radioactive 32P and 35S.
•   Phage contain only DNA and protein, and no other types of molecule.
•   They used phage T2, infecting E. coli.
•   Hershey and Chase showed that 32P-labelled DNA entered the bacterial
    cells when the phage infected them, and that the new generation of phage
    contained a significant amount of that labelled DNA.
•   In contrast, the 35S-labelled protein stayed outside the cells during an
    infection, and none of it ended up in the new phage.
•   This implies that DNA is necessary for phage replication.
              Structure of DNA
• Once the importance of DNA was recognized, it was
  necessary to deduce how the DNA molecule is
  structured. A race between various lab groups ensued,
  and in 1953 James Watson and Francis Crick published
  a model of DNA structure. Their work was based on X-
  ray crystallography data provided by Maurice Wilkins
  and Rosalind Franklin.
• DNA consists of two anti-parallel chains twisted into a
  helix. The nitrogenous bases are paired in the center of
  the molecule, and the phosphate-sugar backbones are
  on the outside.
• Although DNA is the genetic material of all living cells,
  some viruses use RNA as their genetic material.
            Nucleotide Structure
• DNA and RNA are macromolecules composed of subunits called
  nucleotides.
• Each nucleotide of DNA or RNA has 3 parts: a nitrogenous base, a
  sugar, and a phosphate group.
• The phosphate group, PO4, links two sugar molecules in the
  backbone. Each phosphate carries a -1 charge. This causes DNA
  to have an overall negative charge.
• The sugar is ribose in the case of RNA and deoxyribose in the case
  of DNA. has 5 carbons, numbered 1’ through 5’.
   – the nitrogenous base is attached to the 1’ carbon
   – the 2’ carbon has a free -OH group in the case of RNA, but a -H group
     in the case of DNA. The lack of the oxygen atom makes DNA far less
     reactive than RNA.
   – the 3’ carbon has an -OH group on it that links to the phosphate group
     on the next base. The “end” of the DNA molecule is a free 3’ OH group.
   – the 5’ carbon is attached to the phosphate group.
Nucleotide
        More Nucleotide Structure
•   There are 4 possible DNA bases: adenine (A), guanine (G), cytosine (C),
    and thymine (T).
•   Adenine and guanine are purines: they consist of two linked rings of mixed
    nitrogen and carbon atoms.
•   Thymine and cytosine are pyrimidines, which consist of a single ring. In
    RNA, thymine is replaced by uracil (U), which looks like thymine except for
    a single methyl group.
•   Each strand of DNA pairs with a complementary DNA strand. This pairing
    happens because each A is paired with a T, and each G is paired with a C.
    Thus, the information on one DNA strand easily allows the other strand to
    be deduced. The amount of A in DNA always equals the amount of T, and
    the amount of G always equals the amount of C. This is not true in RNA,
    which is usually single-stranded.
•   Pairing is caused by hydrogen bonds, weak links between oxygen and
    nitrogen atoms where one of them has a hydrogen attached.
•   A-T pairs have 2 hydrogen bonds, while G-C pairs have 3 hydrogen bonds.
    G-C pairs are stronger, and they are more frequent in high temperature
    organisms.
Paired Nucleotides
    Semi-conservative Replication
•   Watson and Crick recognized that the
    double stranded DNA molecule could
    replicate by unwinding, then
    synthesizing a new strand for each of
    the old stands.
•   This mode of replication is called
    “semi-conservative”. It means that
    after one DNA molecule has replicated
    to become 2 DNA molecules, each
    new molecule consists of one old
    strand (from the original molecule) and
    one new strand.
•   The information from each old strand
    can be used to create the new strands,
    since A always pairs with T, and G
    always pairs with C.
•   DNA replication starts at specific
    locations “origins of replication”, and
    proceeds in both directions.
           Replication Components
•   The raw materials of DNA synthesis are “nucleoside
    triphosphates”, often written as “dNTPs”.
•   dNTPs have a chain of 3 phosphate groups attached to
    the 5’ carbon of the deoxyribose sugar. Just as with
    ATP, the bonds between the phosphates are high energy
    bonds, and releasing them produces the energy needed
    to drive the synthesis of DNA.
•   Each new nucleotide is added to a growing DNA chain by
    removing the outer 2 phosphates and attaching the
    remaining phosphate to the 3’ OH group of the previous
    nucleotide.
•   The DNA chain is said to grow from 5’ to 3’, which means
    that the first DNA base has a free 5’ end, with attached
    phosphates. The last nucleotide has a free 3’ OH group
    on it. All other bases have their 5’ carbons attached to a
    phosphate, which is attached to the 3’ OH group of the
    previous nucleotide.
•   DNA polymerase is the main enzyme used to replicate
    DNA. However, DNA polymerase is only one enzyme in
    the replication complex. Several other enzymes are
    needed to cause replication to occur.
    Continuous and Discontinuous
             Synthesis
• DNA can only be synthesized from 5’ to 3’, by adding
  new nucleotides to the 3’ end.
• This is a problem, because both strands must be
  synthesized at the replication fork, and one strand will
  necessarily be synthesized in the opposite direction from
  the movement of the replication fork.
• In reality, one strand is synthesized continuously, in the
  same direction that the replication form is moving. This
  is called the “leading strand”.
• The other strand is synthesized in short, discontinuous
  pieces, that are then attached together to form the final
  DNA strand. This is the “lagging strand”. Each fragment
  of the lagging strand is called an “Okazaki fragment”,
  and they are synthesized in the opposite direction that
  the replication fork moves.
          Discontinuous Synthesis
•   Another peculiarity of DNA
    synthesis is that DNA polymerase
    must attach new bases to the 3’
    end of a pre-existing nucleic acid
    chain. All DNA synthesis starts at
    a short double-stranded region.
•   In the cell, short pieces of RNA,
    called “primers” are paired with
    the DNA bases to create to the
    short double stranded regions that
    DNA synthesis builds.
•   The RNA primers are synthesized
    by an enzyme called “primase”,
    and they are removed by DNA
    polymerase during the synthesis
    of the next Okazaki fragment.
•   Joining of the Okazaki fragments
    is done by the enzyme “DNA
    ligase”.
           Replication Miscellany
• Errors occur in DNA replication fairly frequently: the wrong base gets
  inserted due to the peculiarities of nucleotide chemistry. However,
  DNA polymerase has a built-in editing function that removes most of
  the incorrect bases.
• Enzymes that replicate the RNA genomes of some viruses do not
  have editing functions. Thus, mutation rates in RNA viruses are
  100-1000 times higher than in DNA viruses. This rapid mutation
  rate makes it easy for RNA viruses to evade the immune system.

• DNA in cells is “supercoiled”, twisted into tight knots because the
  helix is given a few extra twists beyond what it needs to maintain its
  shape. Supercoiling has the advantage of causing the DNA to be
  more compact inside the cell, but it must be created and maintained
  by various enzymes that wind the DNA, break and rejoin DNA
  strands so they can pass through one another in the winding
  process, and stabilize single stranded molecules.

				
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