LE 16-7
The mechanism of DNA Replication
5 end
Hydrogen bond
3 end
1 nm
3.4 nm
3 end
0.34 nm
Key features of DNA structure
5 end
Partial chemical structure
Space-filling model
When during the cell cycle is DNA synthesized? Draw
The Basic Principle: Base Pairing
• Each strand acts as a template for
building a new strand in replication
• Parent dsDNA molecule unwinds &
base pairs are broken
- two new daughter strands built based on
base-pairing rules
Draw
LE 16-9_1
The parent molecule has
two complementary
strands of DNA. Each base
is paired by hydrogen
bonding with its specific
partner, A with T and G with
C.
LE 16-9_4
A simple model of DNA replication
The first step is separation
of the two parental
DNA strands.
Synthesis of
complementary strands
The nucleotides are
connected to form the
sugar-phosphate backbones of the new strands.
Predicted by Watson and Crick
Semiconservative model of
DNA replication
LE 16-10
Parent cell
Various
proposed
models of
DNA
replication
Conservative
model. The two
parental strands
reassociate after
acting as
templates for
new strands,
thus restoring
the parental
double helix.
Semiconservative
model. The two
strands of the
parental
molecule
separate, and each
functions as a
template for synthesis
of a new, complementary strand.
Dispersive model.
Each strand of
both daughter
molecules
contains
a mixture of
old and newly
synthesized
DNA.
First
replication
Second
replication
• Meselson and Stahl experimentally
supported
one of the replication models
How & which one?
LE 16-11
Heavy
radioisotope
Why label
nitrogen?
Bacteria
cultured in
medium
containing
15N
Bacteria
transferred to
medium
containing
14N
DNA sample
centrifuged
after 20 min
(after first
replication)
DNA sample
centrifuged
after 40 min
(after second
replication)
First replication
Conservative
model
Supported by data
Semiconservative
model
Dispersive
model
Less
dense
More
dense
Second replication
Light
radioisotope
• Replication begins
– at origin of replication (ori)
• Creation of replication bubble with replication forks
at each end (Draw)
•Hundreds to thousands of oris on
eukaryotic chromosome
• Usually one on bacterial chromosome
•Proceeds in both directions from each origin,
until the entire molecule is copied
LE 16-12
Parental (template) strand
Origin of replication
Bubble
Daughter (new) strand
0.25 µm
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). Arrowheads mark replication
forks.
Elongating a New DNA Strand
• Basic components
Template DNA
DNA polymerase
DNA precursors
deoxynucleotide triphosphates
(dATP, dCTP, dGTP,dTTP)
LE 16-13
New strand
5 end
Template strand
3 end
5 end
3 end
Sugar
Base
Phosphate
DNA polymerase
3 end
5’
Pyrophosphate
Nucleoside
triphosphate
5 end
3 end
5 end
Specificity of DNA polymerase
• only adds nucleotides to the free 3hydroxyl
end of dsDNA
• New DNA strand made only in 5’-3’direction
Draw
LE 16-14
3
5
Parental DNA
primer
Leading strand
5
3
Okazaki
fragments
Lagging strand
3
5
DNA pol III
Template
strand
Leading strand
Lagging strand
Template
strand
DNA ligase
Overall direction of replication
LE 16-16
Overall direction of replication
Lagging
Leading
Origin of replication
strand
strand
Lagging
strand
DNA pol III
OVERVIEW
Leading
strand
Leading
strand
5
3
Parental DNA
DNA ligase
Replication fork
Primase
Primer
DNA pol I
DNA pol III Lagging
strand
3
5
Other components of the DNA replication machinery?
DNA helicase- to unwind DNA
Single strand binding proteins- to stabilize ssDNA
DNA ligase- to seal gap in sugar-phosphate backbone (make
phosphodiester bond) between Okazaki fragments
LE 16-15_1
A Closer Look at Lagging Strand Synthesis
3
Primase joins RNA
nucleotides into a primer.
5
5
3
Template
strand
Overall direction of replication
LE 16-15_2
3
Primase joins RNA
nucleotides into a primer.
5
5
Template
strand
3
3
DNA pol III adds
DNA nucleotides to
the primer, forming
an Okazaki fragment.
RNA primer
5
Overall direction of replication
3
5
LE 16-15_3
Primase joins RNA
nucleotides into a primer.
3
5
5
Template
strand
3
3
DNA pol III adds
DNA nucleotides to
the primer, forming
an Okazaki fragment.
RNA primer
3
5
5
After reaching the
next RNA primer (not
shown), DNA pol III
falls off.
Okazaki
fragment
3
3
5
5
Overall direction of replication
LE 16-15_4
Primase joins RNA
nucleotides into a primer.
3
5
5
Template
strand
3
3
DNA pol III adds
DNA nucleotides to
the primer, forming
an Okazaki fragment.
RNA primer
3
5
5
After reaching the
next RNA primer (not
shown), DNA pol III
falls off.
Okazaki
fragment
3
3
5
5
After the second fragment is
primed, DNA pol III adds DNA
nucleotides until it reaches the
first primer and falls off.
5
3
3
5
Overall direction of replication
LE 16-15_5
Primase joins RNA
nucleotides into a primer.
3
5
5
3
Template
strand
3
DNA pol III adds
DNA nucleotides to
the primer, forming
an Okazaki fragment.
RNA primer
3
5
5
After reaching the
next RNA primer (not
shown), DNA pol III
falls off.
Okazaki
fragment
3
3
5
5
After the second fragment is
primed, DNA pol III adds DNA
nucleotides until it reaches the
first primer and falls off.
5
3
3
5
5
3
DNA pol I replaces
the RNA with DNA,
adding to the 3 end
of fragment 2.
3
5
Overall direction of replication
LE 16-15_6
Primase joins RNA
nucleotides into a primer.
3
5
5
3
Template
strand
3
DNA pol III adds
DNA nucleotides to
the primer, forming
an Okazaki fragment.
RNA primer
3
5
5
After reaching the
next RNA primer (not
shown), DNA pol III
falls off.
Okazaki
fragment
3
3
5
5
After the second fragment is
primed, DNA pol III adds DNA
nucleotides until it reaches the
first primer and falls off.
5
3
3
5
5
3
DNA pol I replaces
the RNA with DNA,
adding to the 3 end
of fragment 2.
3
5
DNA ligase forms a
bond between the newest
DNA and the adjacent DNA
of fragment 1.
The lagging
strand in the region
is now complete.
5
3
3
5
Overall direction of replication
Animation: Lagging Strand
Animation: DNA Replication Review
Proofreading and Repairing DNA
•
DNA polymerases proofread
•
Replace mismatched nt in new DNA
• Also
1. Mismatch repair: repair enzymes correct
errors in base pairing
2. Nucleotide excision repair: enzymes cut out
and replace damaged stretches of DNA
Example
DNA exposure to ultraviolet (UV) light
induces chemical crosslinks between
adjacent thymines (thymine dimers)
How to repair?
LE 16-17
A thymine dimer
distorts the DNA molecule.
A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed.
Nuclease
Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA
polymerase
DNA
ligase
DNA ligase seals the
free end of the new DNA
to the old DNA, making the
strand complete.
Is DNA replication of linear chromosomes
ever complete?
Consider the tips (ends) of the leading and
lagging strands.
LE 16-18
5
Leading strand
Lagging strand
End of parental
DNA strands
3
Last fragment
Previous fragment
RNA primer
Lagging strand 5
3
Primer removed but
cannot be replaced
with DNA because
no 3 end available
for DNA polymerase
Removal of primers and
replacement with DNA
where a 3 end is available
5
3
Second round
of replication
5
New leading strand 3
New leading strand 5
3
Further rounds
of replication
Shorter and shorter
daughter molecules
• Ends of eukaryotic chromosomes
– Tipped with many copies of a short DNA repeat
called telomeres (e.g.
human telomere sequence TTAGGG x 100-1,000)
• Added by telomerase , a ribozyme (made of RNA and
proteins)
Function:Telomeres postpone loss of important
genes near ends after each cell division.
Is telomerase found in all eukaryotic cells?
NO, mostly in germ cells but NOT in somatic cells.
What will happen to DNA in cells that continually divide
such as epithelial cells (skin, gut)?
Make a prediction about the length of chromosomes in skin cells
from a 80 year old versus a 4 year old.
Cancer cells are characterized in part by their continuous
cell division. Shouldn’t they ultimately die from loss of genes
due to shortening of chromosomes?
Hypothesize why they continue to divide without injury?
Cancer cells express telomerase, which prevents
chromosome shortening
LE 16-19
Labelled telomeres
Questions?
1 µm