1. History, Structure and Replication
DNA
History, Structure and Replication

2. Mendel and Morgan
Traits were hereditary Traits could be linked Traits were found on chromosomes. QUESTION: What is a chromosome made of? DNA and Proteins

3. The Debate
What was the molecule of heredity? DNA or Protein? Case for Protein: - millions and millions of traits - proteins made of 20 different monomers (amino acids) - greater possibility to code for more traits Case against DNA: - only made of 4 monomers - less possible combinations

4. Fredrick Griffith worked with mice and Streptococcus pneumoniae
bacteria that causes pneumonia
- two strains
1. Rough - no protein coat - lacked gene to make coat
2. Smooth - protein coat - protected bacteria from mouse immune system

5. Injections into mice smooth - death rough - harmless
heated smooth - harmless
heated rough - harmless
heated rough with live smooth - death
heated smooth with live rough - death ????

6. Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they
have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule
and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below:
Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an
unknown, heritable substance from the dead S cells.
EXPERIMENT
RESULTS
CONCLUSION
Living S
(control) cells
Living R
Heat-killed
(control) S cells
Mixture of heat-killed S cells
and living R cells
Mouse dies
Mouse healthy
Living S cells
are found in
blood sample.
Figure 16.2

7. Points of Interest two previous harmless strains combined caused death
when the blood from the dead mouse was examined
found living Smooth
“something” had transformed the rough into smooth
Oswald Avery: found that component to be DNA - not good enough

8. CONFIRMING EVIDENCE Hershey-Chase experiment:
worked with bacteria and a virus known as a bacteriophage:
made of only protein and DNA

10. Phages attach to a bacterium and inject the bacteriophage’s interior substance into the bacterium
caused the bacterium to change and produce more bacteriophages
- the genetic material (protein or DNA) must be injected into the bacterium

11. Hershey and Chase ran two experiments
1. Bacteriophage grown in radioactive sulfur-tagged protein
2. Bacteriophage grown in radioactive phosphorous-tagged DNA

12. IDEA: allow the phages to infect the bacteria and separate the shells from the bacteria
- test the bacteria for radioactivity
- whichever batch was positive identified the genetic material

13. RESULTS:
Radioactive sulfur: (radioactive protein)
bacteria negative for radiation - protein stayed on outside of bacteria
Radioactive phosphorous (radioactive DNA)
bacteria positive for radiation - DNA was injected

14. In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur
and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells.
Radioactivity
(phage protein)
in liquid
Phage
Bacterial cell
Radioactive
protein
Empty
protein shell
DNA
Centrifuge
Pellet (bacterial
cells and contents)
Pellet
Batch 1: Phages were
grown with radioactive
sulfur (35S), which was
incorporated into phage
protein (pink).
Batch 2: Phages were
phosphorus (32P), which
was incorporated into
phage DNA (blue). 1 2 3 4
Agitated in a blender to
separate phages outside
the bacteria from the
bacterial cells.
Mixed radioactively
labeled phages with
bacteria. The phages
infected the bacterial cells.
Centrifuged the mixture
so that bacteria formed
a pellet at the bottom of
the test tube.
Measured the
radioactivity in
the pellet and
the liquid
Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells.
When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus.
Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material.
RESULTS
CONCLUSION
EXPERIMENT
(phage DNA)
in pellet
Figure 16.4

15. Conclusion
DNA injected - DNA caused the change - produced new bacteriophages
DNA IS THE MOLECULE OF HEREDITY

16. Structure of DNA Early discoveries: Erwin Chargaff:
Analyzed the DNA of different species
Known that DNA composed of nucleotides made of three parts
1. Deoxyribose - sugar
2. Phosphate
3. one of 4 different nitrogenous bases
adenine (purine - 2 rings)
thymine (pyrimidine - 1 ring)
guanine (purine - 2 rings)
cytosine (pyrimidine - 1 ring)

17. Chargaff discovered that no matter the source of the DNA
the amount of adenine always equalled the amount of thymine
the amount of guanine always equalled the amount of cytosine
A = T and G = C known as Chargaff’s Rule

18. How was it all put together?
X-ray diffraction photos by Rosalind Franklin - Franklin realized that DNA was composed of two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior
(a) Rosalind Franklin
Franklin’s X-ray diffraction
Photograph of DNA
(b)
Figure 16.6 a, b

19. (a) Key features of DNA structure (c) Space-filling model
Figure 16.7a, c C T A G
0.34 nm
3.4 nm
(a) Key features of DNA structure
1 nm
(c) Space-filling model

20. used Chargaff’s rule and X-ray diffraction photos by R. Franklin
Watson and Crick reasoned that there must be additional specificity of pairing
Dictated by the structure of the bases
used Chargaff’s rule and X-ray diffraction photos by R. Franklin
Once they figured it out, they published a paper and “forgot” to mention Rosalind Franklin

21. CONCLUSION
DNA is a double helix of antiparallel strands with matching purines and pyrimadines
Watson and Crick are RAT BASTARDS
Figure 16.1

22. DNA Structure Phosphate sugar backbone: sides of a ladder
Nitrogenous Base pairs = rungs of the ladder
Twisted Double Helix

23. (b) Partial chemical structure
DNA Structure Strands run anti-parallel: 5` to 3` is countered with 3` to 5` O –O OH
H2C T A C G
CH2 O–
5 end
Hydrogen bond
3 end P
(b) Partial chemical structure
Figure 16.7b

24. Adenine is bonded to thymine
Guanine is bonded to cytosine
- this is why the amounts are always equal
In each pair you have a 2 ring and a 1 ring bonded together
- if had two 2 rings - too wide
- if had two 1 rings - too narrow
one of each - consistent width
WHY A to T and G to C?? and not A to C and G to T??
BONDING of the purines and pyrimidines - PAGE hydrogen bonding has to match up - thymine can only link to adenine

25. N H O
CH3
Sugar
Adenine (A)
Thymine (T)
Guanine (G)
Cytosine (C)
Figure 16.8

26. DNA Replication
Watson and Crick proposed that the double helix would split and act as templates for the building of a new strand
- each resulting strand would be identical
HOWEVER: how did the daughter stand form and how did it relate to the parent DNA?

27. Three Theories of DNA Replication
1. Conservative: Parent splits forms daughter DNA and then comes back together.
- RESULT: Parent DNA strands remain together and form a daughter DNA composed of completely new DNA

28. Three Theories of DNA Replication
2. Semi-conservative: Parent DNA splits and forms daughter DNA -
- each side of the parent DNA acts as a template and is part of the daughter DNA
- RESULT: two strands, each is half parental and half new DNA

29. Three Theories of DNA Replication
3. Dispersive: parent DNA splits apart into pieces and forms daughter DNA
RESULT: Daughter DNA is composed of parent and new
EVIDENCE: Which is correct? Work of Meselson and Stahl

30. 3 Models of Replication Figure 16.10 a–c Parent cell First replication
Conservative
model. The two
parental strands
reassociate
after acting as
templates for
new strands,
thus restoring
the parental
double helix.
Semiconservative
strands of the
parental molecule
separate,
and each functions
as a template
for synthesis of
a new, comple-
mentary strand.
Dispersive
model. Each
strand of both
daughter mol-
ecules contains
a mixture of
old and newly
synthesized
DNA.
Parent cell
First
replication
Second

31. Meselson and Stahl
Bacteria grown in a medium of “heavy” nitrogen (isotope 15N) so the DNA is all heavy
Transferred to a medium of “light” nitrogen (14N) so any new DNA would be lighter than the old
- allowed the DNA to go through one replication and they analyzed the results -
- extracted the DNA and centrifuged it - heavier DNA would sink further in test tube

32. First Round Of Replication
If conservative true: two separate bands after one replication
If semiconservative or dispersive true: get one band after one replication
Actual Results: ONE BAND

33. Figure 16.11
Matthew Meselson and Franklin Stahl cultured E. coli bacteria for several generations
on a medium containing nucleotide precursors labeled with a heavy isotope of nitrogen, 15N. The bacteria
incorporated the heavy nitrogen into their DNA. The scientists then transferred the bacteria to a medium with
only 14N, the lighter, more common isotope of nitrogen. Any new DNA that the bacteria synthesized would be
lighter than the parental DNA made in the 15N medium. Meselson and Stahl could distinguish DNA of different
densities by centrifuging DNA extracted from the bacteria.
EXPERIMENT
The bands in these two centrifuge tubes represent the results of centrifuging two DNA samples from the flask
in step 2, one sample taken after 20 minutes and one after 40 minutes.
RESULTS
Bacteria
cultured in
medium
containing
15N
transferred to
14N 2 1
DNA sample
centrifuged
after 20 min
(after first
replication) 3
after 40 min
(after second 4
Less
dense
More

34. Second Round of Replication
Distinguish between semi-conservative and dispersive
semiconservative - two bands
dispersive - one
Results: two bands = SEMICONSERVATIVE

35. CONCLUSION
Meselson and Stahl concluded that DNA replication follows the semiconservative
model by comparing their result to the results predicted by each of the three models in Figure
The first replication in the 14N medium produced a band of hybrid (15N–14N) DNA. This result eliminated
the conservative model. A second replication produced both light and hybrid DNA, a result that eliminated
the dispersive model and supported the semiconservative model.
First replication
Second replication
Conservative
model
Semiconservative
Dispersive

36. HOW DOES IT ALL WORK???? ENZYMES
Form a Replisome - cluster of proteins and enzymes that form the replication machinery for DNA Replication
Helicase, Topoisomerase, Single Strand Binding Proteins, RNA Primase, DNA Polymerase I and III, Sliding Clamp, Ligase

37. Table 16.1

38. DNA Replication STEP 1: Starting Replication - separating the strands
DNA helicase and gyrase
Gyrase (topoisomerase) - undoes the supercoiling of the DNA so Helicase can break the hydrogen bonds between the strands
-forms a replication bubble and two replication forks
- bubbles eventually merge
- separated strands are held apart by single strand binding proteins

39. Replication Bubbles Figure 16.12 a, b
Replication begins at specific sites
where the two parental strands
separate and form replication
bubbles.
The bubbles expand laterally, as
DNA replication proceeds in both
directions.
Eventually, the replication
bubbles fuse, and synthesis of
the daughter strands is
complete. 1 2 3
Origin of replication
Bubble
Parental (template) strand
Daughter (new) strand
Replication fork
Two daughter DNA molecules
In eukaryotes, DNA replication begins at many sites along the giant
DNA molecule of each chromosome.
In this micrograph, three replication
bubbles are visible along the DNA of
a cultured Chinese hamster cell (TEM).
(b)
(a)
0.25 µm
Figure a, b

40. DNA Replication
STEP 2: Priming the strands: New DNA can’t just start building - it has to attach to an existing chain
- need a PRIMER to get the process started
- a short chain of RNA is formed by PRIMASE - binds to parent DNA strand -

41. DNA Replication STEP 3: Building New DNA
DNA polymerase III - brings in new nucleotides and match them with the correct bases
each nucleotide is a TRIPHOSPHATE like ATP but deoxyribose instead of ribose - gives the energy to build the bonds through dehydration synthesis
- rate of elongation - 50 nucleotide per second in humans
- DNA Pol III is held in place by Sliding Clamp Protein

42. Figure 16.13 New strand Template strand 5 end 3 end Sugar A T Base COH
Pyrophosphate
2 P
Phosphate

43. DNA Replication Effect of ANTIPARALLEL Strands:
Polymerase only adds to the 3` end, not the 5` end
- the chain can only be elongated in the 5` to 3` direction
Results in two strands:
1. Leading strand: moves in the direction of the replication fork - forms one long strand
2. Lagging strand: moves in the opposite direction of the replication fork - forms little strands of DNA called Okazaki fragments
- each fragment requires its own RNA primer

44. Overall direction of replication3 5 1 2
Template strand
RNA primer
Okazaki fragment
Figure 16.15

45. DNA Replication STEP 4: Replacement of RNA primers with DNA
- DNA polymerase I (and possibly another enzyme called RNase H) removes the RNA and replaces it with the appropriate DNA

46. DNA Replication STEP 5: Binding of Okazaki fragments by DNA ligase
DNA Replication Tutorial
DNA Replication Animation
Lagging Strand Detail

47. Overall direction of replication
Figure 16.16
Overall direction of replication
Leading
strand
Lagging
OVERVIEW
Replication fork
DNA pol III
Primase
Primer
DNA pol I
Parental DNA 5 3 4 3 2
Origin of replication
DNA ligase 1
Helicase unwinds the
parental double helix.
Molecules of single-
strand binding protein
stabilize the unwound
template strands.
The leading strand is
synthesized continuously in the
5 3 direction by DNA pol III.
Primase begins synthesis
of RNA primer for fifth
Okazaki fragment.
DNA pol III is completing synthesis of
the fourth fragment, when it reaches the
RNA primer on the third fragment, it will
dissociate, move to the replication fork,
and add DNA nucleotides to the 3 end of the fifth fragment primer. 5
DNA pol I removes the primer from the 5 end
of the second fragment, replacing it with DNA
nucleotides that it adds one by one to the 3 end
of the third fragment. The replacement of the
last RNA nucleotide with DNA leaves the sugar-
phosphate backbone with a free 3 end. 6
DNA ligase bonds
the 3 end of the
second fragment to
the 5 end of the first
fragment. 7

48. DNA Replication Problems
Humans: approximately 3.2 billion nucleotide bases
- bound to have errors - only about 1 every 10 billion
- not a lot but - that is 0.32 errors every replication - could lead to huge problems
DNA has a self correcting enzyme
- proofreads DNA as it is replicated and repairs any damage to DNA
DNA Spell check and autocorrect

49. Replication Repair
TYPES of Repair 1. mismatch repair - wrong nucleotide is put into the DNA - removed and replaced - DNA polymerase II
2. Excision repair - damaged or faulty nucleotides are cut out and replaced by proper ones - NUCLEASE
-DNA constantly is patrolled by these enzymes because it is constantly undergoing damage from outside influences - UV Radiation

50. In nucleotide excision repair
Enzymes cut out and replace damaged stretches of DNA
A thymine dimer
distorts the DNA molecule. 1
A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed. 2
Nuclease
DNA
polymerase 3
Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA
ligase
DNA ligase seals the
Free end of the new DNA
To the old DNA, making the
strand complete. 4
Figure 16.17

51. End of the Line Problems
DNA polymerase can only add on to the 3` end, so on the leading strand the RNA primer leaves a gap
shortens the DNA
- could cause problems
Chromosomes have end caps called telomeres that are redundant non genes
- eventually wear down - possible links to aging

52. Figure 16.18 End of parental DNA strands Leading strand Lagging strand
Last fragment
Previous fragment
RNA primer
Removal of primers and
replacement with DNA
where a 3 end is available
Primer removed but
cannot be replaced
with DNA because
no 3 end available
for DNA polymerase
Second round
of replication
New leading strand
New lagging strand 5
Further rounds
Shorter and shorter
daughter molecules 5 3

53. If the chromosomes of germ cells became shorter in every cell cycle essential genes would eventually be missing from the gametes they produce
An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells

54. Differences in Replication in Prokaryotes
Bacterial Chromosomes are Circular - no ends - no need for telomeres