INCLUDING STRUCTURE

1
DNA AS THE GENETIC MATERIAL

• INCLUDING STRUCTURE REPLICATION

2
GENETIC MATERIAL

• Frederick Griffith (1928)
• English bacteriologist
• Experiments indicated that DNA is the genetic
  material
• Most thought that protein was a more likely
  candidate
• Why do you think this might be?
• Used Streptococcus pneumoniae
• a.k.a., Diplococcus pneumoniae
• Pneumonia-causing bacterium

3
GENETIC MATERIAL

• Frederick Griffith (1928)
• Streptococcus pneumoniae
• Two strains
• S strain
• Smooth colonies (macroscopic)
• Pathogenic
• R strain
• Rough colonies (macroscopic)
• Non-pathogenic

4
GENETIC MATERIAL

• Frederick Griffith (1928)
• R-strain bacteria mouse ? alive
• S-strain bacteria mouse ? dead
• Heat-killed S-strain bacteria mouse ? alive
• Conclusions?

5
GENETIC MATERIAL

• Frederick Griffith (1928)
• Heat-killed S live R mouse ? dead mouse!!!
• Live S cells present in blood
• R transformed into S
• Bacterial transformation

How?
6
GENETIC MATERIAL

• Frederick Griffith (1928)
• Bacterial transformation
• A chemical substance was transferred from the
  dead S bacteria to the live R bacteria
• What is this substance?
• Why is DNA a more likely candidate than protein?
• Most of the world still thought that protein was
  the genetic material

7
GENETIC MATERIAL

• Oswald Avery (1944)
• American bacteriologist
• Furthered Griffiths experiments
• Determined that the transforming substance was
  DNA
• Thus, DNA is the genetic material
• People still not convinced

8
GENETIC MATERIAL

• Oswald Avery (1944)
• Purified various macromolecules from S bacteria
• DNA
• RNA
• Protein
• Sugars
• Attempted to transform R bacteria with each

9
GENETIC MATERIAL

• Oswald Avery (1944)
• S sugars live R ? no live S
• S protein live R ? no live S
• S RNA live R ? no live S
• S DNA live R ?live S!!! (dead mouse)
• DNA is the genetic material!
• People still not convinced

10
GENETIC MATERIAL

• Oswald Avery (1944)
• Critics of Avery
• DNA was contaminated with small amounts of
  protein
• Enzymes are active in small amounts
• Protein contaminants in the DNA preparation are
  the transforming material
• (Wrong, but rational)

11
GENETIC MATERIAL

• Oswald Avery (1944)
• Response to critics
• S DNA live R ?live S
• Include protease ?live S!!!
• Protein enzymatically degraded
• Include DNAse ?no live S!!!
• DNA enzymatically degraded
• DNA is the genetic material
• People still not convinced (Thick skulls, I guess)

12
GENETIC MATERIAL

• Hershey Chase (1952)
• Established that DNA is the genetic material
• Used T2 bacteriophage
• Virus infecting bacteria
• Composed of DNA protein
• Reprograms infected cell
• Reprogramming requires genetic material

13
GENETIC MATERIAL

• Hershey Chase (1952)
• Radiolabeled phage with 35S
• Labels protein, not DNA
• Radiolabeled phage with 32P
• Labels DNA, not protein

14
GENETIC MATERIAL

• Hershey Chase (1952)
• Infect E coli with 35S-labeled phage
• Disrupt, centrifuge
• Supernatant hot
• Pellet cold
• Protein does not enter cell

15
GENETIC MATERIAL

• Hershey Chase (1952)
• Infect E coli with 32P-labeled phage
• Disrupt, centrifuge
• Supernatant cold, pellet hot
• DNA enters cell
• DNA is the genetic material, and people are
  finally convinced!!!

16
DNASTRUCTURE
17
DNA STRUCTURE

• Crick Watson (1953)
• Determined the 3-D structure of DNA
• Structure of single strand already known

Single strand of DNA
18
DNA STRUCTURE

• Crick Watson (1953)
• Relied heavily on work by
• Erwin Chargaff
• Rosalind Franklin
• Maurice Wilkins
• How many experiments do you imagine Crick
  Watson performed?
• Who shared the Nobel in 1962 with Crick Watson?

19
DNA STRUCTURE

• Crick Watson (1953)
• Chargaffs Rules
• For any organisms DNA
• A T
• G C
• Crick Watsons realization
• This indicates a specific relationship between A
  T, and between G C

Erwin Chargaff
20
DNA STRUCTURE

• Crick Watson (1953)
• Maurice Wilkins Rosalind Franklin
• X-ray crystallography
• DNA is helical
• Full turn every 3.4 nanometers
• Diameter of helix is 2 nanometers

21
DNA STRUCTURE

• Franklins data and Chargaffs data were critical
  in allowing Crick Watson to determine the
  three-dimensional structure of DNA

22
DNA DOUBLE HELIX
5'
3'
3'
5'
Antiparallel
23
DNA DOUBLE HELIX

• 3 and 5 ends of DNA strands refer to the carbon
  not attached to an adjacent nucleotide

24
DNA DOUBLE HELIX

• A T are held together by two H-bonds
• G C are held together by three H-bonds

25
DNA REPLICATION
26
DNA REPLICATION

• Crick Watson (1954)
• The second DNA strand is repetitious redundant
• Why are there two strands?
• Important for replication
• Semiconservative replication

27
DNA REPLICATION

• Begins at origins of replication
• Specific DNA sequences
• One (prokaryotes) or several (eukaryotes)
• Replication bubbles formed, enlarged, fused
• Entire DNA molecule ultimately replicated

28
DNA REPLICATION

• Various enzymes and other proteins are involved
  in DNA replication
• Helicase
• Single-strand binding proteins
• Primase
• DNA polymerase
• DNA ligase

29
DNA REPLICATION

• Helicase unwinds the DNA double helix
• Strands are separated (unzipped)
• Begins at origin of replication
• Continue as replication forks progress
• Single-strand binding proteins bind to the
  separated DNA strands
• Attachment to unpaired single strands prevents
  reannealing to their complementary strand

30
DNA REPLICATION

• DNA polymerase cannot initiate a polynucleotide
  strand
• It can add to the 3 end of an existing strand
• Primase forms a short RNA primer
• Complementary to a region of ssDNA
• Primase can initiate a polynucleotide strand
• (Primase is an RNA polymerase)

31
DNA REPLICATION

• DNA polymerase extends this RNA polymer
• Deoxynucleotides added to the 3 end
• 5 ? 3 synthesis

32
DNA REPLICATION

• DNA polymerase can only add to the 3 end of a
  polynucleotide
• One strand is synthesized continuously
• Leading strand
• One strand is synthesized discontinuously
• Lagging strand

Replication Fork
33
DNA REPLICATION

• DNA polymerase ultimately removes the short RNA
  primers
• Replaced with DNA
• DNA ligase covalently links the discontinuously
  synthesized DNA fragments
• Okazaki fragments

Replication Fork
34
DNA REPLICATION
35
DNA REPLICATION
36
DNA REPLICATION

• Energy requirements
• Deoxynucleotides supplied as triphosphates
• Two phosphates removed
• Breaking of phosphate bond releases energy

37
DNA REPLICATION

• What are the functions of these enzymes?
• Helicase
• Single-strand binding proteins
• Primase
• DNA polymerase
• DNA ligase

38
MUTATION

• DNA replication is very accurate
• Specificity defined by base pairing
• DNA polymerase does make mistakes
• 1/10,000 base pairs
• Try doing thousands of first grade math problems
• These errors are termed mutations
• Most of these errors are corrected

39
MUTATION

• Proofreading
• DNA polymerase proofreads shortly after addition
• Incorrectly paired nucleotides excised, replaced
• Still, it doesnt correct everything
• Mismatch repair
• Specific enzymes fix incorrectly paired
  nucleotides
• Other systems
• Also involved in correcting incorrectly
  synthesized or damaged DNA

40
MUTATION

• The initial mutation rate is 1/10,000
• Following correction, the ultimate mutation rate
  is 1/billion
• Mutations are rare events that are vitally
  important in the history of life
• Ultimate source for all genetic variation
• Individuals with beneficial variations are more
  likely to survive, reproduce, and pass these
  genetic traits to the next generation
• Natural selection

41
MUTATION

• Reverse transcriptase is an enzyme possessed by
  retroviruses (including HIV)
• Synthesizes DNA using an RNA template
• Initial mutation rate 1/10,000
• Lacks proofreading ability
• Ultimate mutation rate 1/10,000
• HIV and other retroviruses display very high
  mutation rates

42
MUTATION

• HIV and other retroviruses display very high
  mutation rates
• Its all about RT (reverse transcriptase)
• Consequence of lack of DNA repair
• Results in very high genetic variation within
  viral populations
• Some fraction of this population is always
  unrecognizable by the hosts immune system
• HIV evolves faster than the immune system can
  mount a response