2. 2
History of DNA
• Early scientists thought protein was
the cell’s hereditary material because
it was more complex than DNA
• Proteins were composed of 20
different amino acids in long
polypeptide chains
3. 3
The Genetic Material
Frederick Griffith, 1928
studied Streptococcus pneumoniae, a
pathogenic bacterium causing pneumonia
there are 2 strains of Streptococcus:
- S strain is virulent
- R strain is nonvirulent
Griffith infected mice with these strains
hoping to understand the difference
between the strains
4. 4
Griffith’s results:
- live S strain cells killed the mice
- live R strain cells did not kill the mice
- heat-killed S strain cells did not kill
the mice
- heat-killed S strain + live R strain
cells killed the mice
6. 6
Griffith’s conclusion:
- information specifying virulence passed
from the dead S strain cells into the live R
strain cells
- Griffith called the transfer of this information
transformation
7. 7
The Genetic Material
Avery, MacLeod, & McCarty, 1944
repeated Griffith’s experiment using purified
cell extracts and discovered:
- removal of all protein from the
transforming material did not destroy its
ability to transform R strain cells
- DNA-digesting enzymes destroyed all
transforming ability
- the transforming material is DNA
8. 8
The Genetic Material
Hershey & Chase, 1952
- investigated bacteriophages: viruses that
infect bacteria
- the bacteriophage was composed of only
DNA and protein
- they wanted to determine which of these
molecules is the genetic material that is
injected into the bacteria
9. 9
The Genetic Material
- Bacteriophage DNA was labeled with
radioactive phosphorus (32P)
- Bacteriophage protein was labeled with
radioactive sulfur (35S)
- radioactive molecules were tracked
- only the bacteriophage DNA (as indicated
by the 32P) entered the bacteria and was
used to produce more bacteriophage
- conclusion: DNA is the genetic material
11. 11
DNA Structure
DNA is a nucleic acid.
The building blocks of DNA are
nucleotides, each composed of:
–a 5-carbon sugar called deoxyribose
–a phosphate group (PO4)
–a nitrogenous base
• adenine, thymine, cytosine, guanine
13. 13
DNA Structure
The nucleotide structure consists of
–the nitrogenous base attached to the 1’
carbon of deoxyribose
–the phosphate group attached to the 5’
carbon of deoxyribose
–a free hydroxyl group (-OH) at the 3’
carbon of deoxyribose
15. 15
DNA Structure
Nucleotides are connected to each other to
form a long chain
phosphodiester bond: bond between
adjacent nucleotides
–formed between the phosphate group of
one nucleotide and the 3’ –OH of the
next nucleotide
The chain of nucleotides has a 5’ to 3’
orientation.
17. 17
DNA Structure
Determining the 3-dimmensional structure of
DNA involved the work of a few scientists:
–Erwin Chargaff determined that
• amount of adenine = amount of thymine
• amount of cytosine = amount of guanine
This is known as Chargaff’s Rules
18. 18
DNA Structure
Rosalind Franklin and Maurice Wilkins
–Franklin performed X-ray diffraction
studies to identify the 3-D structure
–discovered that DNA is helical
–discovered that the molecule has a
diameter of 2nm and makes a complete
turn of the helix every 3.4 nm
19. 19
James Watson and Francis Crick, 1953
– deduced the structure of DNA using evidence
from Chargaff, Franklin, and others
– proposed a double helix structure
The double helix consists of:
– 2 sugar-phosphate backbones
– nitrogenous bases toward the interior of the
molecule
– bases form hydrogen bonds with
complementary bases on the opposite
sugar-phosphate backbone
20. 20
DNA Structure
The two strands of nucleotides are
antiparallel to each other
–one is oriented 5’ to 3’, the other 3’ to 5’
The two strands wrap around each other to
create the helical shape of the molecule.
22. 22
Replication Facts
• DNA has to be copied before a cell divides
• DNA is copied during the S or synthesis
phase of interphase
• New cells will need identical DNA strands
23. 23
Synthesis Phase (S phase)
• S phase during interphase of the cell cycle
• Nucleus of eukaryotes
Mitosis
-prophase
-metaphase
-anaphase
-telophase
G1 G2
S
phase
interphase
DNA replication takes
place in the S phase.
24. 24
DNA Replication
Matthew Meselson & Franklin Stahl, 1958
investigated the process of DNA replication
considered 3 possible mechanisms:
– conservative model
– semiconservative model
– dispersive model
26. 28
DNA Replication
Meselson and Stahl concluded that the
mechanism of DNA replication is the
semiconservative model.
Each strand of DNA acts as a template for
the synthesis of a new strand.
27. 29
•DNA replication is the process of copying a DNA molecule. Replication is
semiconservative, with each strand of the original double helix (parental molecule)
serving as a template (mold or model) for a new strand in a daughter molecule. This
process consists of:
•Unwinding (initiation): old strands of the parent DNA molecule are unwound as
weak hydrogen bonds between the paired bases are “unzipped” and broken by the
enzyme helicase.
•Complementary base pairing (elongation): free nucleotides present in the nucleus
bind with complementary bases on unzipped portions of the two strands of DNA; this
process is catalyzed by DNA polymerase.
•Joining (elongation): complementary nucleotides bond to each other to form new
strands; each daughter DNA molecule contains an old strand and a new strand; this
process is also catalyzed by DNA polymerase.
•Termination – replication is terminated differently in prokaryotes and eukaryotes
Ends in prokaryotes when origin is reached
Ends in eukaryotes when telomere is reached
telomeres – repeated DNA sequence on the ends of eukaryotic
chromosomes
•DNA replication must occur before a cell can divide; in cancer, drugs with molecules
similar to the four nucleotides are used to stop replication.
DNA Replication
29. How is a Repl. origin selected?
Priming at the oriC (Bacterial)
Origin
Initiation
30. + Hu on the origin
+ ATP
Ready to bind primase!
31. 1. Many copies of dnaA bind the four 9-mers; DNA wraps
around dnaA forming “Initial Complex”. This requires ATP
and a protein Hu that is already bound to the DNA.
3. Two copies of dnaB (helicase) bind the 13-mers. This
requires dnaC (which does not remain with the
Prepriming Complex) and ATP.
4. Primase binds to dnaB (helicase) and the DNA.
2. This triggers opening of the 13-mers (Open complex).
5. dnaB:primase complex moves along the template 3’>5’
synthesizing RNA primers 5’>3’ for Pol III to extend.
Order of events at OriC
33. Enzymes Involved in
Elongation:
1. DNA-dependent DNA polymerases
– synthesize DNA from dNTPs
– require a template strand and a primer strand with
a 3’-OH end
– all synthesize from 5’ to 3’ (add nt to 3’ end only)
36. Proofreading Activity
Insertion of the wrong nucleotide causes the DNA
polymerase to stall, and then the 3’-to-5’ exonuclease
activity removes the mispaired A nt. The polymerase then
continues adding nts to the primer.
37. If DNA polymerases only synthesize 5’ to 3’, how
does the replication fork move directionally?
38. • Lagging strand synthesized as small (~100-1000 bp)
fragments - “Okazaki fragments” .
• Okazaki fragments begin as very short 6-15 nt RNA
primers synthesized by primase.
2. Primase - RNA polymerase that synthesizes the
RNA primers (11-12 nt that start with pppAG) for both
lagging and leading strand synthesis
40. Pol III extends the RNA primers until the 3’ end of an
Okazaki fragment reaches the 5’ end of a downstream
Okazaki fragment.
Lagging strand synthesis (continued)
Then, Pol I degrades the RNA part with its 5’-3’
exonuclease activity, and replaces it with DNA. Pol I
is not highly processive, so stops before going far.
41. At this stage, Lagging strand is a series of DNA
fragments (without gaps).
Fragments stitched together covalently by DNA
Ligase.
3. DNA Ligase - joins the 5’ phosphate of one DNA
molecule to the 3’ OH of another, using energy in the
form of NAD (prokaryotes) or ATP (eukaryotes). It
prefers substrates that are double-stranded, with only
one strand needing ligation, and lacking gaps.
42. Ligase will join these two G--G--A--T--C--C--T--T--G--A--T--C--C
| | | | | | | | | | | | |
C--C--T--A--G G--A--A--C--T--A--G--G
Ligase will NOT join these
two.
G--G--A--T--C--C--T--T--G--A--T--C--C
| | | | | | | | | | | |
C--C--T--A--G C--A--A--C--T--A--G--G
Ligase will NOT join these
two.
G--G--A--T--C--C--T--T--G--A--T--C--C
| | | | | | | | | | | |
C--C--T--A--A G--A--A--C--T--A--G--G
Ligase will NOT join these
two.
G--G--A--T--C--C--T--T--G--A--T--C--C
| | | | | | | | | | | |
C--C--T--A--G G--T--A--C--T--A--G--G
Ligase will NOT join these
two. C--C--T--A--G C--T--A--C--T--A--G--G
DNA Ligase Substrate Specificity
43. HO
P
3'
5'
2
1
+ AMP
3'
P
AMP
P
AMP
+
HO
3'
P
5'
Ligase
NAD
1 2
1
3'
NMN
P
Ligase
NAD NMN
+AMP
Mechanism of
Prokaryotic
DNA Ligase-
Ligase binds NAD,
cleaves it, leaving AMP
attached to it.
Ligase-AMP binds and
attaches to 5’ end of a
DNA molecule (1) via the
AMP.
The DNA fragment with
the 3’ OH end (2) reacts
with the phosphodiester,
displacing the AMP-
ligase.
(Eukaryotic
DNA ligase
uses ATP
as AMP
donor,
instead of
NAD).
44. Replisome - DNA and protein machinery at a
replication fork.
Other proteins needed for DNA replication:
4. DNA Helicase (dnaB gene) – hexameric protein,
unwinds DNA strands, uses ATP.
5. SSB – single-strand DNA binding protein, prevents
strands from re-annealing and from being degraded,
stimulates DNA Pol III.
6. Gyrase – Topoisomerase II, keeps DNA ahead of
fork from over winding (i.e., relieves torsional strain).
46. Rubber Band Model
of Supercoiling DNA
DNA Gyrase relaxes positive
supercoils by breaking and
rejoining both DNA strands.
47. 5'
3'
3'
5'
5'
5'
DNA Polymerase III
actshere
DNA Polymerase I extends
one Okazaki fragment and
removes the RNA from
another.
DNA Ligase then joins
fragmentstogether.
ssDNA binding
protein(SSB)
Helicase (DnaB)
Primase
3'
Gyrase
Sp in n in g
at 10,000
r p m
A p ro k ary o tic fo rk is trav ellin g at 50 to 100 k b / m in u te.
Eu k ary o tic fo rk s trav el at 0.5 - 5 k b / m in u te.
Primosome
A Replisome
48. What about the ends (or telomeres) of linear chromosomes?
DNA polymerase/ligase cannot fill gap at end of chromosome after RNA
primer is removed. this gap is not filled, chromosomes would
become shorter each round of replication!
Solution:
1. Eukaryotes have tandemly repeated sequences at the ends of their
chromosomes.
2. Telomerase (composed of protein and RNA complementary to the
telomere repeat) binds to the terminal telomere repeat and
catalyzes the addition of of new repeats.
3. Compensates by lengthening the chromosome.
4. Absence or mutation of telomerase activity results in chromosome
shortening and limited cell division.
50. Final Step - Assembly into Nucleosomes:
• As DNA unwinds, nucleosomes must disassemble.
• Histones and the associated chromatin proteins must be duplicated
by new protein synthesis.
• Newly replicated DNA is assembled into nucleosomes almost
immediately.
• Histone chaperone proteins control the assembly.
Fig. 3.17
51. 53
Proofreading New DNA
• DNA polymerase initially makes about 1 in
10,000 base pairing errors
• Enzymes proofread and correct these
mistakes
• The new error rate for DNA that has been
proofread is 1 in 1 billion base pairing
errors
53. •Prokaryotic Replication
•Bacteria have a single loop of DNA that must
replicate before the cell divides.
•Replication in prokaryotes may be bidirectional
from one point of origin or in only one direction.
•Replication only proceeds in one direction, from 5'
to 3'.
•Bacterial cells are able to replicate their DNA at a
rate of about 106 base pairs per minute.
•Bacterial cells can complete DNA replication in 40
minutes; eukaryotes take hours.
54. 56
•Eukaryotic Replication
•Replication in eukaryotes starts at many
points of origin and spreads with many
replication bubbles—places where the DNA
strands are separating and replication is
occurring.
•Replication forks are the V-shape ends of
the replication bubbles; the sites of DNA
replication.
•Eukaryotes replicate their DNA at a slower
rate – 500 to 5,000 base pairs per minute.
•Eukaryotes take hours to complete DNA
replication.
58. 60
•Replication Errors
•A genetic mutation is a permanent change in the sequence
of bases.
•Base changes during replication are one way mutations
occur.
•A mismatched nucleotide may occur once per 100,000
base pairs, causing a pause in replication.
•Proofreading is the removal of a mismatched nucleotide;
DNA repair enzymes perform this proofreading function
and reduce the error rate to one per billion base pairs.
•Incorrect base pairs that survive the proofreading process
contribute to gene mutations.
