LE 16-7

1
LE 16-7
The mechanism of DNA Replication
5? end
Hydrogen bond
3? end
1 nm
3.4 nm
3? end
0.34 nm
5? end
Space-filling model
Partial chemical structure
Key features of DNA structure
When during the cell cycle is DNA synthesized?
Draw
2
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
3
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.
4
LE 16-9_4
A simple model of DNA replication
The nucleotides are connected to form the
sugar-phosphate back- bones of the new strands.
The first step is separation of the two
parental DNA strands.
Synthesis of complementary strands
Predicted by Watson and Crick Semiconservative
model of DNA replication
5
LE 16-10
First replication
Second replication
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,
comple-mentary strand.
Dispersive model. Each strand of both daughter
molecules contains a mixture of old and newly
synthesized DNA.
6

• Meselson and Stahl experimentally supported
• one of the replication models
• How which one?

7
LE 16-11
Bacteria cultured in medium containing 15N
Bacteria transferred to medium containing 14N
Heavy radioisotope
Light radioisotope
Why label nitrogen?
DNA sample centrifuged after 20 min (after
first replication)
DNA sample centrifuged after 40 min (after
second replication)
Less dense
More dense
First replication
Second replication
Conservative model
Supported by data
Semiconservative model
Dispersive model
8

• 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

9
LE 16-12
Parental (template) strand
0.25 µm
Origin of replication
Daughter (new) strand
Replication fork
Bubble
Two daughter DNA molecules
In this micrograph, three replication bubbles are
visible along the DNA of a cultured Chinese
hamster cell (TEM). Arrowheads mark replication
forks.
In eukaryotes, DNA replication begins at many
sites along the giant DNA molecule of each
chromosome.
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Elongating a New DNA Strand

• Basic components
• Template DNA
• DNA polymerase
• DNA precursors
• deoxynucleotide triphosphates
• (dATP, dCTP, dGTP,dTTP)

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LE 16-13
New strand
Template strand
3 end
5 end
5 end
3 end
Sugar
Base
Phosphate
DNA polymerase
3 end
5
3 end
Pyrophosphate
Nucleoside triphosphate
5 end
5 end
12
Specificity of DNA polymerase

• only adds nucleotides to the free 3??hydroxyl end
  of dsDNA
• New DNA strand made only in 5-3direction

Draw
13
LE 16-14
3
5
primer
Parental DNA
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
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LE 16-16
Overall direction of replication
Leading strand
Lagging strand
Origin of replication
Lagging strand
Leading strand
OVERVIEW
DNA pol III
Leading strand
DNA ligase
Replication fork
5
DNA pol I
3
Primase
Lagging strand
Parental DNA
DNA pol III
Primer
3
5
15
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
16
LE 16-15_1
A Closer Look at Lagging Strand Synthesis
Primase joins RNA nucleotides into a primer.
3
5
3?
5
Template strand
Overall direction of replication
17
LE 16-15_2
Primase joins RNA nucleotides into a primer.
3
5
3?
5
Template strand
DNA pol III adds DNA nucleotides to the
primer, forming an Okazaki fragment.
3
5
RNA primer
3
5
Overall direction of replication
18
LE 16-15_3
Primase joins RNA nucleotides into a primer.
3
5
3?
5
Template strand
DNA pol III adds DNA nucleotides to the
primer, forming an Okazaki fragment.
3
5
RNA primer
3
5
After reaching the next RNA primer
(not shown), DNA pol III falls off.
Okazaki fragment
3
3
5
5
Overall direction of replication
19
LE 16-15_4
Primase joins RNA nucleotides into a primer.
3
5
3?
5
Template strand
DNA pol III adds DNA nucleotides to the
primer, forming an Okazaki fragment.
3
5
RNA primer
3
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
20
LE 16-15_5
Primase joins RNA nucleotides into a primer.
3
5
3?
5
Template strand
DNA pol III adds DNA nucleotides to the
primer, forming an Okazaki fragment.
3
5
RNA primer
3
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
DNA pol I replaces the RNA with
DNA, adding to the 3 end of fragment 2.
5
3
3
5
Overall direction of replication
21
LE 16-15_6
Primase joins RNA nucleotides into a primer.
3
5
3?
5
Template strand
DNA pol III adds DNA nucleotides to the
primer, forming an Okazaki fragment.
3
5
RNA primer
3
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
DNA pol I replaces the RNA with
DNA, adding to the 3 end of fragment 2.
5
3
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
22
Animation Lagging Strand
23
Animation DNA Replication Review
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Proofreading and Repairing DNA

• DNA polymerases proofread
• Replace mismatched nt in new DNA
• Also
• Mismatch repair repair enzymes correct errors in
  base pairing
• 2. Nucleotide excision repair enzymes cut out
  and replace damaged stretches of DNA

25
Example DNA exposure to ultraviolet (UV)
light induces chemical crosslinks between
adjacent thymines (thymine dimers)
How to repair?
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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.
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Is DNA replication of linear chromosomes ever
complete? Consider the tips (ends) of the
leading and lagging strands.
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LE 16-18
5
Leading strand
End of parental DNA strands
Lagging strand
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
3
New leading strand
5
New leading strand
3
Further rounds of replication
Shorter and shorter daughter molecules
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• 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)
• FunctionTelomeres 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.
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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. Shouldnt 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
31
LE 16-19
Labelled telomeres
Questions?
1 µm