The Structure, Replication and Repair of DNA

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Chapter 10
The Structure, Replication and Repair of DNA
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Key Questions

• How did biologists discover what genes were made
  of and what they did?
• What is the structure of DNA?
• How does DNAs structure allow it to act as a
  template for its own replications?
• What is a mutation and why are mutations
  important?

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Structure of DNA-Overview

• Each nucleotide of DNA consists of
• A sugar deoxyribose
• A phosphate
• A base there are 4 bases
• 2 of the 4 bases are pyrimidines cytosine (C)
  and thymine (T)
• The other 2 bases are purines denine (A) and
  guanine (G)

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Structure of DNA-A Historic Story (1)

• Late 1800s scientists postulated a biochemical
  basis
• Friedrich Miescher (1869) isolated DNA (called
  it nucleic acid)
• Researchers became convinced chromosomes carry
  genetic information
• 1920s to 1940s expected the protein portion of
  chromosomes to be the genetic material
• Late 1920s Frederick Griffith was working with
  Streptococcus pneumoniae?????
• Strains that secrete capsules look smooth and can
  cause fatal infections in mice
• Strains that do not secrete capsules look rough
  and infections are not fatal in mice
• Griffiths experiments (next page) showed that
• Genetic material from the heat-killed type S
  bacteria had been transferred to the living type
  R bacteria
• This trait gave them the capsule and was passed
  on to their offspring
• Griffith did not know the biochemical basis of
  his transforming principle

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Griffiths Bacterial Transformations

• Rough strains (R) without capsule are not fatal
• No living bacteria found in blood
• Smooth strains (S) with capsule are fatal
• Capsule prevents immune system from killing
  bacteria
• Living bacteria found in blood
• If mice are injected with heat-killed type S,
  they survive
• Mixing live R with heat-killed S kills the mouse
• Blood contains living S bacteria
• Transformation

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Structure of DNA-A Historic Story (2)

• Avery, MacLeod, and McCarty used purification
  methods to reveal that DNA is the genetic
  material
• 1940s interested in bacterial transformation
• Only purified DNA from type S could transform
  type R
• Purified DNA might still contain traces of
  contamination that may be the transforming
  principle
• Added DNase, RNase and proteases
• RNase and protease had no effect
• With DNase no transformation
• DNA is the genetic material
• 1952, Hershey and Chase studied T2 virus
  infecting Escherichia coli
• Bacteriophage or phage
• Phage coat made entirely of protein
• DNA found inside capsid

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Structure of DNA-A Historic Story (3)

• 1952, Hershey and Chase studied T2 virus
  infecting Escherichia coli
• Bacteriophage or phage
• Phage coat made entirely of protein
• Bacteriophage
• A virus that infects bacteria
• Viruses are composed of protein and DNA (or RNA)
• Viruses are capable of forcing host cells to make
  more viruses
• Viruses are not living organisms

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Structure of DNA-A Historic Story (3)

• Shearing force from a blender will separate the
  phage coat from the bacteria
• 35S will label proteins only 32P will label DNA
  only
• Experiment to find what is injected into
  bacteria- DNA or protein?
• DNA found inside capsid Results support DNA as
  the genetic material

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Hershey and Chase The Blender Guy/Gal
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Structure of DNA-A Historic Story (3)
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Structure of DNA-A Historic Story (4)

• Chagaffs Rules
• Found the proportions of the bases in many
  different species
• The amount of A is equal to T
• The amount of G is equal to C

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Structure of DNA-A Historic Story (5)

• Solving DNA structure
• 1953, James Watson and Francis Crick, with
  Maurice Wilkins, proposed the structure of the
  DNA double helix
• Watson and Crick used Linus Paulings method of
  working out protein structures using simple
  ball-and-stick models
• Rosalind Franklins X-ray diffraction results
  provided crucial information
• Erwin Chargoff analyzed base composition of DNA
  that also provided important information

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Rosalind Franklins Contribution

• Crystallographer
• Accurately measured the density of DNA, the
  number of water molecules
• Discovered that DNA had 2 slightly different
  structures

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Structure of DNA-A Historic Story (5)

• Used Franklins data and work extensively, but
  did not cite it
• Applied Chargaffs Rules
• Built several models of DNA
• Found ball-and-stick model consistent with data
• Watson and Crick awarded Nobel Prize in 1962
• Rosalind Franklin had died and the Nobel is not
  awarded posthumously

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DNA Structure Linkages

• DNA is
• Double stranded
• Helical
• Sugar-phosphate backbone
• Bases on the inside
• Stabilized by hydrogen bonding
• Base pairs with specific pairing
• AT/GC or Chargoffs rule
• A pairs with T
• G pairs with C
• Keeps with consistent
• 10 base pairs per turn
• 2 DNA strands are complementary
• 5 GCGGATTT 3
• 3 CGCCTAAA 5
• 2 strands are antiparallel
• One strand 5 to 3
• Other stand 3 to 5

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Computer Generated Model of DNA
Figure 10-9
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What Is a Gene?

• To Mendel, in 1865, it was just an abstraction
• Places on chromosomes
• Pure information
• Important to understand genes in chemical terms
• 1908, Archbold Garrod proposed relationship
  between genes and the production of enzymes
• Studied patients with metabolic defects
• Alkaptonuria- patients body accumulates abnormal
  levels of homogentisic acid (alkapton)
• Hypothesized disease due to missing enzyme
• Knew it had a recessive pattern of inheritance
• Inborn error of metabolism

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What Causes Alkaptonuria?
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Biochemical Importance of Genes

• Alkaptonuria black urine stains
• Garrod suspected that it might be inherited
• Caused by a recessive allele
• A crucial enzyme is missing HA accumulates in
  the body and is excreted in the urine
• Suggested that genes might work by specifying
  enzymes

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1 Gene 1 Enzyme

• In the early 1940s, George Beadle and Edward
  Tatum rediscovered Garrods work, using
  Neurospora crassa (common bread mold), showed
  that 1 gene could specify 1 enzyme
• Minimum requirements for growth are carbon source
  (sugar), inorganic salts, and biotin
• Mutant strains would be unable to grow unless
  supplemented
• Compare to wild-type or normal
• A single mutation resulted in the requirement for
  a single type of vitamin
• Stimulated research into other substances
  including arginine, an amino acid

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1 Gene 1 Enzyme

• Isolated several mutants requiring arginine for
  growth
• Examined for ability to grow in the presence of
  precursors
• 3 groups based on requirements
• Beadle and Tatum conclude that single gene
  controls the synthesis of a single enzyme
• One gene one enzyme hypothesis

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1 Gene 1 Polypeptide

• Sickle Cell Anemia
• Differences in gene has caused differences in the
  hemoglobin protein, not in the enzyme
• One gene one enzyme hypothesis has been
  modified
• Enzymes are only one category of cellular
  proteins
• More accurate to say one gene encodes a
  polypeptide
• Hemoglobin composed of 4 polypeptides required
  for function
• One gene one polypeptide theory
• Genes influence phenotype by specifying
  polypeptides

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DNA Replication

• 3 different models for DNA replication proposed
  in late 1950s
• Semiconservative
• Conservative
• Dispersive
• Newly made strands are daughter strands
• Original strands are parental strands

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DNA Replication

• In 1958, Matthew Meselson and Franklin Stahl
  devised experiment to differentiate among 3
  proposed mechanisms
• Nitrogen comes in a common light form (14N) and a
  rare heavy form (15N)
• Grew E.coli in medium with only 15N
• Then switched to medium with only 14N
• Collected sample after each generation
• Original parental strands would be 15N while
  newly made strands would be 14N
• Results consistent with semiconservative mechanism

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Simultaneously Copying DNA Strands

• DNA replication occurs in replication forks
• These are Y-shaped regions of DNA where the 2
  strands of the helix have come apart
• Nucleotides add directly to the 3 end of an RNA
  primer the other strand is produced in short
  fragments (Okazaki Fragments) that are joined
  together by DNA ligase
• In the leading strand
• DNA primase makes one RNA primer
• DNA polymerase attaches nucleotides in a 5 to 3
  direction as it slides forward
• In the lagging strand
• DNA synthesized 5 to 3 but in a direction away
  from the fork
• Okazaki fragments made as a short RNA primer made
  by DNA primase at the 5 end and then DNA laid
  down by DNA polymerase
• RNA primers will be removed by DNA polymerase and
  filled in with DNA
• DNA ligase will join adjacent DNA fragments

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DNA Polymerization

• DNA polymerase enzyme that strings together the
  nucleotides
• During replication 2 parental strands separate
  and serve as template strands
• New nucleotides must obey the AT/GC rule
• End result 2 new double helices with same base
  sequence as original

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DNA Polymerization
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RNA Polymerase

• An RNA polymerase makes an RNA primer that
  provides a 3 end for DNA polymerase
• Then DNA polymerase links together the
  nucleotides that assemble opposite the template
  into a new strand of DNA

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Sources of Genetic Diversity

• Recombination
• Crossing over
• Mixing of gametes
• Mutations permanent changes in DNA probably
  the ultimate source
• Kinds of Mutations
• Point mutations change 1 or several nucleotide
  pairs
• Base substitution replacement of 1 base by
  another
• Insertion addition of 1 or more nucleotides
• Deletion the removal of 1 or more nucleotides
• Chromosomal mutations large regions of
  chromosome changes
• Deficiencies
• Translocations
• Inversion
• Duplications

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Mutation Rate

• Measure of how often mutations occur
• Depends on how often a sequence mutates, and on
  how efficiently cells repair these mutations
• Mutation hot spots exist that are more likely to
  mutate
• Mutation rates are usually low

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Sunburn Damages DNA
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Correcting Mistakes

• An enzyme detects something wrong in 1 strand of
  the DNA and removes it
• Then DNA polymerase copies the information in the
  intact second strand and creates a new stretch of
  DNA
• DNA ligase seals the gap