DNA Structure and Replication

1
DNA Structure and Replication
2
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 DNAs central role came in
  the 1950s, but important experiments were done
  earlier.

3
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.

4
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.

5
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.

6
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.

7
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.

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Nucleotide
9
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.

10
Paired Nucleotides
11
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.

12
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.

13
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.

14
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.

15
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.