Transcript Foundations of Biology - Geoscience Research Institute

Isaiah 40:28
28 Hast thou not known? hast
thou not heard, that the
everlasting God, the LORD,
the Creator of the ends of the
earth, fainteth not, neither is
weary? there is no searching
of his understanding.
©2001 Timothy G. Standish
Replication
Timothy G. Standish, Ph. D.
©2001 Timothy G. Standish
The Information Catch-22
“With only poor copying fidelity, a primitive
system could carry little genetic information
without L [the mutation rate] becoming
unbearably large, and how a primitive system
could then improve its fidelity and also
evolve into a sexual system with crossover
beggars the imagination.”
Hoyle F. 1987. "Mathematics of Evolution",
Acorn Enterprises: Memphis TN, 1999, p20
©2001 Timothy G. Standish
Tools of Replication
Enzymes are the tools of replication:
DNA Polymerase - Matches the
correct nucleotides then joins adjacent
nucleotides to each other
Primase - Provides an RNA primer to
start polymerization
Ligase - Joins adjacent DNA strands
together (fixes “nicks”)
©2001 Timothy G. Standish
More Tools of Replication
Helicase - Unwinds the DNA and melts it
Single-Strand Binding Proteins - Keep
the DNA single stranded after it has been
melted by helicase
Gyrase - A topisomerase that relieves
torsional strain in the DNA molecule
Telomerase - Finishes off the ends of
DNA strands
©2001 Timothy G. Standish
Extension - The Replication Fork
5’
3’
3’
5’
3’
5’
5’
3’
5’
Primase
- Makes
RNA
primers
Lagging Strand
Okazaki
fragment
5’
RNA
Primers
3’
5’
Single-strand
binding
proteins Prevent DNA
from reanealing
DNA
Polymerase
Leading Strand
5’
3’
5’
3’
Helicase Melts DNA
Gyrase Relieves
torsional strain
©2001 Timothy G. Standish
Extension - Okazaki Fragments
5’
3’
Okazaki Fragment
DNA
Pol.
3’
5’
RNA Primer
DNA Polymerase has 5’ to 3’ exonuclease activity.
When it sees an RNA/DNA hybrid, it chops out the
RNA and some DNA in the 5’ to 3’ direction.
5’
3’
DNA
Pol.
RNA and DNA Fragments
3’
5’
RNA Primer
DNA Polymerase falls off leaving a nick.
5’
3’
Ligase
3’
5’
RNA Primer
Nick
The nick is removed when
DNA ligase joins (ligates) the
DNA fragments.
©2001 Timothy G. Standish
The Role of DNA Gyrase
Helicase
©2001 Timothy G. Standish
The Role of DNA Gyrase
Supercoiled DNA
Gyrase
Helicase
©2001 Timothy G. Standish
The Role of DNA Gyrase
Gyrase
©2001 Timothy G. Standish
The Role of DNA Gyrase
Gyrase
©2001 Timothy G. Standish
The Role of DNA Gyrase
Gyrase
©2001 Timothy G. Standish
The Role of DNA Gyrase
Gyrase
©2001 Timothy G. Standish
The Role of DNA Gyrase
Gyrase
©2001 Timothy G. Standish
The Role of DNA Gyrase
Gyrase
©2001 Timothy G. Standish
The Role of DNA Gyrase
Gyrase
©2001 Timothy G. Standish
The Role of DNA Gyrase
Gyrase
©2001 Timothy G. Standish
The Role of DNA Gyrase
Gyrase
©2001 Timothy G. Standish
E. coli DNA Polymerases
E. coli has three identified DNA polymerases each of
which has significantly different physical characteristics
and roles in the cell
Polymerase
I
II
III
5’- 3’ Polymerization
3’-5’ Exonuclease
5’-3’ Exonulcease
Molecules/cell
Major function
Yes
Yes
Yes
Yes
Yes
Yes
Yes
400
Proofreading/
Removal of
RNA primers
109,000 Daltons
No
?
Repair of
damaged
DNA
No
15
Replication
polymerization
10 subunits
600,000 Daltons
Klenow fragment (76,000 Daltons),
prepared by mild proteolysis, lacks
5’ to 3’ exonuclease activity and is
used in sequencing
©2001 Timothy G. Standish
Telomerase
At the end of linear chromosomes the lagging strand can’t be
completed as the last primer is removed and no 3’ hydroxyl
group is available for DNA polymerase to extend from
Telomere
5’
3’
3’
5’
Degradation of RNA primer at the 5’ end
5’
3’
3’
5’
Next replication
5’
3’
3’
5’
+
3’
5’
5’
3’
©2001 Timothy G. Standish
Telomerase
Telomerase is a ribo-protein complex that adds
nucleotides to the end of chromosomes thus restoring
their length
Telomerase
5’GACCGAGCCTCTTGGGTTGGGGTTG
3’CTGGCTCGG
AACCCCAAC
RNA
©2001 Timothy G. Standish
Telomerase
Telomerase is a ribo-protein complex that adds
nucleotides to the end of chromosomes thus restoring
their length
Telomerase
5’GACCGAGCCTCTTGGGTTGGGGTTGGGGTTG
3’CTGGCTCGG
AACCCCAAC
RNA
©2001 Timothy G. Standish
Telomerase
Telomerase is a ribo-protein complex that adds
nucleotides to the end of chromosomes thus restoring
their length
Telomerase
5’GACCGAGCCTCTTGGGTTGGGGTTGGGGTTGGGGTTG
3’CTGGCTCGG
AACCCCAAC
RNA
©2001 Timothy G. Standish
Telomerase
The TTGGGG repeating telomere sequence can form
a hairpin due to unusual GG base pairing
5’GACCGAGCCTCTTGGGTTGGGGTTGGGGTTGGGGTTG
3’CTGGCTCGG
Guanine
Guanine
©2001 Timothy G. Standish
Telomerase
The TTGGGG repeating telomere sequence can form
a hairpin due to unusual GG base pairing
5’GACCGAGCCTCTTGGGTTGGGGTTGGGG
DNA
T GGGGTTG
3’GTTGGGG T
Pol.
3’CTGGCTCGG
©2001 Timothy G. Standish
Telomerase
The TTGGGG repeating telomere sequence can form
a hairpin due to unusual GG base pairing
Endo5’GACCGAGCCTCTTGGGTTGGGGTTGGGG
DNA
T
nuclease
T
AGAACCCAACCCGTTGGGG
Pol.
3’CTGGCTCGG
©2001 Timothy G. Standish
Telomerase
The TTGGGG repeating telomere sequence can form
a hairpin due to unusual GG base pairing
5’GACCGAGCCTCTTGGGTTGGG
3’CTGGCTCGGAGAACCCAACCC
Endonuclease
©2001 Timothy G. Standish
©2001 Timothy G. Standish
Mutation
When Mistakes Are Made
5’
DNA
Pol.
5’
5’
DNA
Pol.
3’ to 5’ Exonuclease activity
5’
3’
DNA
Pol.
3’
5’
3’
5’
©2001 Timothy G. Standish
Mutation
Excision Repair
5’
3’
3’
5’
5’
3’
3’
EndoNuclease
5’
©2001 Timothy G. Standish
Mutation
Excision Repair
5’
3’
3’
5’
5’
3’
3’
5’
3’
EndoNuclease
5’
Nicks
DNA
Pol.
3’
5’
©2001 Timothy G. Standish
Mutation
Excision Repair
5’
3’
3’
5’
5’
3’
3’
5’
3’
EndoNuclease
5’
DNA
Pol.
3’
5’
©2001 Timothy G. Standish
Mutation
Excision Repair
5’
3’
3’
5’
5’
3’
3’
EndoNuclease
5’
Nicks
5’
3’
Ligase
3’
5’
Nick
©2001 Timothy G. Standish
O
N
N
N
N
H
N
H
H
©2001 Timothy G. Standish
DNA Replication:
How We Know
There are three ways in which DNA could be replicated:
Conservative - Old
Semi-conservative Old strands serve as
templates for new
strands resulting in
double-stranded
DNA made of both
old and new strands
double-stranded
DNA serves as a
template for two
new strands which
then join together,
giving two old
strands together
and two new
Dispersive - In
strands together
which sections of
the old strands are
dispersed in the
new strands
+
+ or
+
+
©2001 Timothy G. Standish
The Meselson-Stahl
Experiment
OH
N
The Meselson-Stahl experiment
N
N
N N
demonstrated that replication is
semiconservative
OH H
This experiment took advantage of
the fact that nucleotide bases contain nitrogen
Thus DNA contains nitrogen
The most common form of Nitrogen is N14 with 7
protons and 7 neutrons
N15 is called “heavy nitrogen” as it has 8 neutrons
thus increasing its mass by 1 atomic mass unit
HO P
O
H2
O
©2001 Timothy G. Standish
The Meselson-Stahl
Experiment
Transfer to
normal N14
media
Bacteria grown in
N15 media for
several replications
The conservative and
dispersive models
make predictions that
do not come true thus,
buy deduction, the
semiconservative
model must be true.
After 20 min.
(1 replication)
transfer DNA
to centrifuge
tube and
centrifuge
Prediction after
2 or more
replications
X
X X
©2001 Timothy G. Standish
The Current Eukaryotic
Recombination Model
Homologous chromosomes
Meiosis Prophase I
©2001 Timothy G. Standish
The Current Eukaryotic
Recombination Model
Exonuclease
Double strand break
©2001 Timothy G. Standish
The Current Eukaryotic
Recombination Model
Exonuclease
©2001 Timothy G. Standish
The Current Eukaryotic
Recombination Model
Exonuclease
©2001 Timothy G. Standish
The Current Eukaryotic
Recombination Model
Exonuclease
©2001 Timothy G. Standish
The Current Eukaryotic
Recombination Model
DNA
Polymerase
©2001 Timothy G. Standish
The Current Eukaryotic
Recombination Model
DNA
Polymerase
©2001 Timothy G. Standish
The Current Eukaryotic
Recombination Model
DNA
Polymerase
©2001 Timothy G. Standish
The Current Eukaryotic
Recombination Model
DNA
Polymerase
©2001 Timothy G. Standish
The Current Eukaryotic
Recombination Model
©2001 Timothy G. Standish
Holliday Structure
©2001 Timothy G. Standish
Holliday Structure
Bend
©2001 Timothy G. Standish
Holliday Structure
Bend
Twist
©2001 Timothy G. Standish
Holliday Structure
Cut
©2001 Timothy G. Standish
Holliday Structure
Cut
©2001 Timothy G. Standish
Holliday Structure
Cut
©2001 Timothy G. Standish
Holliday Structure
Cut
©2001 Timothy G. Standish
Holliday Structure
©2001 Timothy G. Standish
Cutting The Holliday Structure
©2001 Timothy G. Standish