DNA

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DNA

• the discovery
• DNA structure
• DNA replication

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Discovery of DNA as genetic material

• 1920s scientists new that chromosomes were made
  up of DNA and protein
• techniques used a red-dye that binds
  specifically to DNA
• found that DNA was stored in the nucleus
• amount of DNA varied by species
• gametes contained ½ the amount of DNA as somatic
  cells

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Discovery of DNA as genetic material

• Still begged the question
• ? What is the hereditary material the DNA or
  the protein?
• Two sets of experiments answered this question
  one involving bacteria the other involving
  viruses

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Discovery of DNA as genetic material

• Frederick Griffith (1920s) an English physician
• worked with Streptococcus pneumonia, a bacteria
  that causes pneumonia in humans
• two forms of the bacteria
• virulent form S form
• avirulent form R form

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Discovery of DNA as genetic material

• S form
• contains a polysaccharide capsule
• gives bacterial colonies a smooth appearance
• capsule protects the bacterial cell from a
  persons immune system ? virulence factor

• R form
• does not contain a polysaccharide capsule
• has a rough appearance
• susceptible to attack by the immune system

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Discovery of DNA as genetic material

• Griffiths purpose was to develop a vaccine
  against the Streptococcus pneumoniae
• he inoculated some mice with heat-killed S strain
  ? no infection
• he infected some mice with a combination of
  heat-killed S strain and R strain ? mice died of
  pneumonia
• blood of mice full of virulent, S strain bacteria

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Discovery of DNA as genetic material

• Griffiths conclusion
• Some of the R strain bacteria had become
  transformed by the heat-killed S strain into
  virulent S strain bacteria.

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Discovery of DNA as genetic material

• Griffiths conclusion did not answer the
  questions of what was genetic material
• it just established the notion that some
  substance existed the chemical transforming
  principle

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Discovery of DNA as genetic material

• Oswald Avery
• treated samples known to contain pneumoccal
  transforming principle in a variety of ways to
  destroy different types of molecules (proteins,
  nucleic acids, carbohydrates, and lipids)
• Results If DNA in a sample was destroyed,
  transforming activity was lost but there was no
  loss of activity if one of the other organic
  compounds was destroyed.

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Discovery of DNA as genetic material

• Avery and his colleagues published their findings
  in 1944, but results were not seriously
  considered by the scientific community
• most scientists did not believe DNA was
  chemically complex enough to be genetic material
  (as compared to proteins)
• bacterial genetics was a new field of study did
  bacteria even contain genes?

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Discovery of DNA as genetic material

• Studies using viral DNA (Alfred Hershey Martha
  Chase- 1952)
• Hershey-Chase experiment
• involved a bacteriophage T2
• virus that infects bacteria
• consists of DNA contained within a protein coat

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Discovery of DNA as genetic material

• T2 bacteriophage lifecycle
• bacteriophage attacks bacterial cell
• only one portion enters the cell (DNA or
  protein?)
• 20 minutes later bacterial cell bursts releasing
  dozens of new viruses
• Bacterial cell gets converted into a
  bacteriophage producing factory.

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Discovery of DNA as genetic material

• Hershey-Chase experiment
• traced the protein and DNA components of the T2
  bacteriophage by radioactively labeling the
  following portions
• In amino acids cysteine methionine, sulfur is
  a component ? S35
• In DNA, phosphate is present ? P32

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Discovery of DNA as genetic material

• Hershey Chase allowed either the P32 or S35
  labeled viruses to attach to the bacteria
• After a few minutes, the mixtures were agitated
  which stripped away the parts of the virus that
  were not attached separated this from the
  bacteria
• Result Most of the S35 labeled portion of the
  bacteriophages seperated from the bacteria

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Discovery of DNA as genetic material

• Hershey-Chase Experiment Conclusions
• DNA was transferred into the bacteria ?
• DNA is the carrier of genetic information

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Structure of DNA

• Things to consider for scientists
• How is DNA replicated?
• How does DNA cause the synthesis of specific
  proteins?

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Structure of DNA- its characterization

• 1950s Rosalind Franklin attempted to visualize
  the structure of DNA via X-ray crystallographs
• Revealed a helical structure

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Structure of DNA- its characterization

• Erwin Chargaff (1950) found that DNA from many
  different species have equal amounts of the
  nitrogen bases adenine and thymine (AT), and
  guanine and cytosine (GC).
• Chargaffs rule the abundance of purines (AG)
  equals the amount of pyrimidines (TC)

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Structure of DNA- its characterization

• James Watson Francis Crick (1953) built a
  model out of tin that established the general
  structure of DNA
• Looked at X-ray crystallography results
  determined helical nature, as well as, distances
  within the helix
• Results of previous density measurements
  suggested that DNA contained 2 strands
• Previous modeling studies also suggested that the
  2 strands run in opposite directions
  (antiparallel)

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DNA structure- 5 points

• DNA is a double-stranded helix
• DNA has a uniform diameter
• DNA has a right-handed twist
• DNA has antiparallel sides
• The sugar-phosphate backbone coil around the
  outside of the helix, and the nitrogenous bases
  point toward the center.

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DNA structure- nitrogenous bases

• Two strands of DNA are held together by hydrogen
  bonds between the nitrogenous bases
• Adenine (A) pairs with thiamine (T) forming two
  hydrogen bonds
• Guanine (G) pairs with cytosine (C) by forming
  three hydrogen bonds
• Complementary Base Pairing

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DNA structure- antiparallel sides

• Direction of the polynucleotide can be defined by
  the direction of the phosphodiester linkages
  between adjacent nucleotides
• diester two bonds formed by OH groups on the
  deoxyribose and phosphate groups
• notice the position of the 3 and 5 carbons

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DNA structure- antiparallel sides

• the two ends of the DNA chain differ
• one end is a free 5 phosphate group 5end
• other end is a free 3 hydroxyl group (-OH) 3
  end

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DNA structure essential to function

• DNA structure proposed by Watson Crick accounts
  for important functions

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DNA structure essential to function

• the nitrogenous base sequence accounts for an
  organisms genetic information ? genetic
  variation
• genetic material is susceptible to mutation by
  altering of nitrogenous base sequence
• genetic material can be precisely replicated due
  to complementary base pairing
• nitrogenous base sequence can be expressed as
  phenotypes in organisms (DNA ? RNA ? protein)

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How does DNA replicate?

• Watson-Crick model of DNA suggested that the
  basis for copying genetic information is
  complimentary
• If
• 5 ATTGCAT- 3
• Then the partner sequence is
• 3 TAACGTA- 5

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How does DNA replicate?

• occurs in two steps
• 1- DNA is unwound to separate the two template
  strands
• 2- new nucleotides are linked by covalent
  bonding to each new strand in a complementary
  sequence to the old strands

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Semiconservative replication

• the two copies of DNA that result from
  replication each contain
• One newly formed strand of DNA and one old
  strand of DNA

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How does DNA replicate?

• new nucleotides are always added to the 3 end of
  DNA
• there is a free OH group that can react with the
  triphosphate end (or 5 end) of the new
  nucleotide
• two template strands are replicated, then, in
  opposite directions
• involves many enzymes, but DNA polymerase is the
  most noteworthy (adds new nucleotides to growing
  strands)

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FIG. 11.10
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The replication complex

• this is a large protein complex that causes the
  interaction of the template DNA and the enzymes
  involved in replication
• replication complexes attach to various origins
  of replication in the DNA at the same time
• DNA replicates in both directions from the point
  of origin, creating two replication forks

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The replication complex

• Because the DNA strands are antiparalell, the new
  DNA is made in opposite directions
• DNA going toward the replication fork is made by
  continuously adding new nucleotides (leading
  strand)
• DNA being made away from the replication fork
  (lagging strand) is synthesized in short segments
  (Okazaki fragments) and are later connected by
  the enzyme ligase .

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FIG. 11.11
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The replication complex

• the replication complexes stay stationary, while
  the template DNA strands move through them

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Role of DNA polymerase

• job is to attach new nucleotides to the 3
  growing end of new DNA strands
• require a RNA primer to attach to DNA (made by
  primase)
• 3 main protein subunits
• - large a subunit attaches nucleotides in the
  5? 3 direction
• - smaller e subunit that proofreads newly formed
  DNA
• - b2 subunit that clamps the enzyme to the
  template strand

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Role of DNA polymerase

• enzyme threads the DNA through the replication
  complex at a rapid rate
• approx. 1000 nucleotides/second

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Steps to DNA replication

• 1. DNA helicase (enzyme) unwinds the DNA. The
  junction between the unwound part and the open
  part is called a replication fork.
• 2. DNA polymerase adds the complementary
  nucleotides and binds the sugars and phosphates.
  DNA polymerase travels from the 3' to the 5' end.

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Steps to DNA replication

• 3. DNA polymerase adds complementary nucleotides
  on the other side of the ladder. Traveling in the
  opposite direction.
• 4. One side is the leading strand - it follows
  the helicase as it unwinds.
• 5. The other side is the lagging strand - its
  moving away from the helicase

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Steps to DNA replication

• Problem it reaches the replication fork, but the
  helicase is moving in the opposite direction. It
  stops, and another polymerase binds farther down
  the chain.
• This process creates several fragments, called
  Okazaki Fragments, that are bound together by DNA
  ligase.

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Steps to DNA replication

• 6. During replication, there are many points
  along the DNA that are synthesized at the same
  time (multiple replication forks). It would take
  forever to go from one end to the other, it is
  more efficient to open up several points at one
  time.

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An animation

• http//www.stolaf.edu/people/giannini/flashanimat/
  molgenetics/dna-rna2.swf

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DNA proofreading and repair

• not doing this can have a big price
• the incorrect transfer of genetic information to
  new cells

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3 DNA repair systems

• 1- proofreading corrects errors in replication
  as DNA polymerase makes new strands
• 2- mismatch repair DNA is scanned immediately
  after it is made and base-pairing mismatches are
  corrected
• 3- excision repair abnormal N- bases are removed
  due to chemical damage, and replace with
  functional N- bases.