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Transcript of ©2001 Timothy G. Standish Isaiah 40:28 28Hast thou not known? hast thou not heard, that the...
![Page 1: ©2001 Timothy G. Standish Isaiah 40:28 28Hast thou not known? hast thou not heard, that the everlasting God, the LORD, the Creator of the ends of the earth,](https://reader036.fdocuments.us/reader036/viewer/2022062322/56649f4f5503460f94c712e0/html5/thumbnails/1.jpg)
©2001 Timothy G. Standish
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.
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©2001 Timothy G. Standish
ReplicationReplicationTimothy G. Standish, Ph. D.
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©2001 Timothy G. Standish
The Information Catch 22The Information Catch 22With 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., "Mathematics of Evolution", [1987], Acorn Enterprises: Memphis TN, 1999, p20
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©2001 Timothy G. Standish
Tools of ReplicationTools of ReplicationEnzymes 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”)
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©2001 Timothy G. Standish
More Tools of ReplicationMore Tools of ReplicationHelicase - Unwinds the DNA and melts itSingle 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
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©2001 Timothy G. Standish
Leading StrandLeading Strand
Laging StrandLaging Strand
3’
5’3’
5’
Extension - The Replication ForkExtension - The Replication Fork5’
5’5’3’
3’
5’3’3’
5’
Single strand binding proteins - Prevent DNA from re-anealing
DNA Polymerase
Okazaki fragment
RNA Primers
Primase - Makes RNA primers
5’3’
5’
Gyrase - Relieves torsional strain
Helicase - Melts DNA
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©2001 Timothy G. Standish
Extension - Okazaki FragmentsExtension - Okazaki Fragments
The nick is removed when DNA ligase joins (ligates) the DNA fragments.
3’ 5’5’ 3’
RNA PrimerOkazaki Fragment
RNA and DNA Fragments
Nick
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.
DNA Polymerase falls off leaving a nick.
DNAPol.
3’ 5’5’ 3’
RNA Primer
DNAPol.
3’ 5’5’ 3’
RNA PrimerLigase
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©2001 Timothy G. Standish
The Role of DNA GyraseThe Role of DNA Gyrase
Helicase
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©2001 Timothy G. Standish
The Role of DNA GyraseThe Role of DNA Gyrase
Helicase
Supercoiled DNA
Gyrase
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©2001 Timothy G. Standish
The Role of DNA GyraseThe Role of DNA Gyrase
Gyrase
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©2001 Timothy G. Standish
The Role of DNA GyraseThe Role of DNA Gyrase
Gyrase
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©2001 Timothy G. Standish
The Role of DNA GyraseThe Role of DNA Gyrase
Gyrase
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©2001 Timothy G. Standish
The Role of DNA GyraseThe Role of DNA Gyrase
Gyrase
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©2001 Timothy G. Standish
The Role of DNA GyraseThe Role of DNA Gyrase
Gyrase
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©2001 Timothy G. Standish
The Role of DNA GyraseThe Role of DNA Gyrase
Gyrase
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©2001 Timothy G. Standish
The Role of DNA GyraseThe Role of DNA Gyrase
Gyrase
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©2001 Timothy G. Standish
The Role of DNA GyraseThe Role of DNA Gyrase
Gyrase
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©2001 Timothy G. Standish
The Role of DNA GyraseThe Role of DNA Gyrase
Gyrase
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©2001 Timothy G. Standish
E. coliE. coli DNA Polymerases DNA Polymerases E. coli has three identified DNA polymerases each of
which has significantly different physical characteristics and roles in the cell
Replication polymerization
10 subunits 600,000 Daltons
II IIIIPolymerase
Major function400 ? 15Molecules/cell
Yes Yes Yes5’- 3’ Polymerization
Yes Yes Yes3’-5’ Exonuclease
Klenow fragment (76,000 Daltons), prepared by mild proteolysis, lacks 5’ to 3’ exonuclease activity and is
used in sequencing
Repair of damaged
DNA
Yes No No5’-3’ Exonulcease
Proofreading/ Removal of
RNA primers109,000 Daltons
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©2001 Timothy G. Standish
Telomere
TelomeraseTelomeraseAt 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
3’5’5’3’
+
3’5’5’3’
Degradation of RNA primer at the 5’ end
3’5’
5’3’
3’5’5’3’
Next replication
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©2001 Timothy G. Standish
AACCCCAAC
TelomeraseTelomerase
RNA
TelomeraseTelomeraseTelomerase is a ribo-protein complex that adds nucleotides to the end of chromosomes thus restoring their length
GGGTTG5’GACCGAGCCTCTTGGGTTG3’CTGGCTCGG
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©2001 Timothy G. Standish
AACCCCAAC
TelomeraseTelomerase
RNA
TelomeraseTelomeraseTelomerase is a ribo-protein complex that adds nucleotides to the end of chromosomes thus restoring their length
5’GACCGAGCCTCTTGGGTTG3’CTGGCTCGG
GGGTTGGGGTTG
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©2001 Timothy G. Standish
AACCCCAAC
TelomeraseTelomerase
RNA
TelomeraseTelomeraseTelomerase is a ribo-protein complex that adds nucleotides to the end of chromosomes thus restoring their length
5’GACCGAGCCTCTTGGGTTG3’CTGGCTCGG
GGGTTG GGGTTGGGGTTG
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©2001 Timothy G. Standish
TelomeraseTelomeraseThe TTGGGG repeating telomere sequence can form a hairpin due to unusual GG base pairing
5’GACCGAGCCTCTTGGGTTGGGGTTGGGGTTGGGGTTG3’CTGGCTCGG
O
N
HNH
H
N
N
N
Guanine
O
N
HN H
H
N
N
NGuanine
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©2001 Timothy G. Standish
TelomeraseTelomeraseThe TTGGGG repeating telomere sequence can form a hairpin due to unusual GG base pairing
5’GACCGAGCCTCTTGGGTTGGGGTTGGGG3’GTTGGGG3’CTGGCTCGG
TTGGGGTTGDNA
Pol.
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©2001 Timothy G. Standish
TelomeraseTelomeraseThe TTGGGG repeating telomere sequence can form a hairpin due to unusual GG base pairing
5’GACCGAGCCTCTTGGGTTGGGGTTGGGGAGAACCCAACCCGTTGGGG3’CTGGCTCGG
TT
DNAPol.
Endo-nuclease
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©2001 Timothy G. Standish
TelomeraseTelomeraseThe TTGGGG repeating telomere sequence can form a hairpin due to unusual GG base pairing
5’GACCGAGCCTCTTGGGTTGGG3’CTGGCTCGG
Endo-nuclease
AGAACCCAACCCGTTGGGGT
T
GTTGGGG
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©2001 Timothy G. Standish
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©2001 Timothy G. Standish
MutationMutationWhen Mistakes Are MadeWhen Mistakes Are Made
5’ 3’
5’
DNAPol.
5’
5’ 3’
5’ 3’
5’
DNAPol.
DNAPol.
Mism
atch
3’ to 5’ Exonuclease activity
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©2001 Timothy G. Standish
Thim
ine
Dimer
MutationMutationExcision RepairExcision Repair
3’
5’ 3’
5’
5’ 3’
3’ 5’
Endo-Nuclease
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©2001 Timothy G. Standish
5’ 3’
3’ 5’
5’ 3’
3’ 5’
MutationMutationExcision RepairExcision Repair
3’
5’ 3’
5’
Endo-Nuclease
NicksDNAPol.
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©2001 Timothy G. Standish
5’ 3’
3’ 5’
MutationMutationExcision RepairExcision Repair
3’
5’ 3’
5’
5’ 3’
3’ 5’
DNAPol.
Endo-Nuclease
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©2001 Timothy G. Standish
5’ 3’
3’ 5’
5’ 3’
3’ 5’
MutationMutationExcision RepairExcision Repair
3’
5’ 3’
5’
DNAPol.
Ligase
Endo-Nuclease
Nicks
Nick
Ligase
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©2001 Timothy G. Standish
O
N
H
N
H
H
N
N
N
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©2001 Timothy G. Standish
DNA Replication:DNA Replication:How We KnowHow We Know
There are three ways in which DNA could be replicated:
+
NewOld
+
Old
N
ewOld
N
ew
OldConservative - Old double stranded DNA serves as a template for two new strands which then join together, giving two old strands together and two new strands together
OldSemi-conservative - Old strands serve as templates for new strands resulting in double stranded DNA made of both old and new strands
Old
Dispersive - In which sections of the old strands are dispersed in the new strands
+
Old +
N
ewOld +
N
ew
+
Old +
N
ewOld +
N
ew
or
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©2001 Timothy G. Standish
The Meselson-Stahl The Meselson-Stahl ExperimentExperiment
The Meselson-Stahl experiment demonstrated that replication is semiconservative
This experiment took advantage of the fact that nucleotide bases contain nitrogen
Thus DNA contains nitrogen
OH
HOH
P
O
HO ONH2
N N
N N
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
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©2001 Timothy G. Standish
After 20 min. (1 replication) transfer DNA to centrifuge tube and centrifuge
Disper
sive m
odel
predict
ion
Conservativ
e
model pre
diction
Semi-c
onservativ
e
model pre
diction
The Meselson-Stahl The Meselson-Stahl ExperimentExperiment
Prediction after 2 or more replications
Bacteria grown in N15 media for several replications
Transfer to normal N14 media
X
X
XThe conservative and dispersive models make predictions that do not come true thus, buy deduction, the semi-conservative model must be true.
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©2001 Timothy G. Standish
The Current Eukaryotic The Current Eukaryotic Recombination ModelRecombination Model
Meiosis Prophase I
Homologous chromosomes
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©2001 Timothy G. Standish
The Current Eukaryotic The Current Eukaryotic Recombination ModelRecombination Model
Double strand break
Exo-nuclease
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©2001 Timothy G. Standish
The Current Eukaryotic The Current Eukaryotic Recombination ModelRecombination Model
Exo-nuclease
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©2001 Timothy G. Standish
The Current Eukaryotic The Current Eukaryotic Recombination ModelRecombination Model
Exo-nuclease
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©2001 Timothy G. Standish
The Current Eukaryotic The Current Eukaryotic Recombination ModelRecombination Model
Exo-nuclease
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©2001 Timothy G. Standish
The Current Eukaryotic The Current Eukaryotic Recombination ModelRecombination Model
DNAPolymerase
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©2001 Timothy G. Standish
The Current Eukaryotic The Current Eukaryotic Recombination ModelRecombination Model
DNAPolymerase
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©2001 Timothy G. Standish
The Current Eukaryotic The Current Eukaryotic Recombination ModelRecombination Model
DNAPolymerase
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©2001 Timothy G. Standish
The Current Eukaryotic The Current Eukaryotic Recombination ModelRecombination Model
DNAPolymerase
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©2001 Timothy G. Standish
The Current Eukaryotic The Current Eukaryotic Recombination ModelRecombination Model
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©2001 Timothy G. Standish
Holliday StructureHolliday Structure
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©2001 Timothy G. Standish
Holliday StructureHolliday Structure
Bend
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©2001 Timothy G. Standish
Holliday StructureHolliday Structure
Bend
Twist
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©2001 Timothy G. Standish
Holliday StructureHolliday Structure
Cut
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©2001 Timothy G. Standish
Holliday StructureHolliday Structure
Cut
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©2001 Timothy G. Standish
Holliday StructureHolliday Structure
Cut
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©2001 Timothy G. Standish
Holliday StructureHolliday Structure
Cut
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©2001 Timothy G. Standish
Holliday StructureHolliday Structure
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©2001 Timothy G. Standish
Cutting The Holliday StructureCutting The Holliday Structure