For quite some time, scientists have been interested in chromosomes WHY???
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For quite some time, scientists have been interested in chromosomes
• WHY???
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Chromosomes
• They replicate prior to both mitosis and meiosis? How?
• They carry information for genetic traits (genotype determines phenotype). How?
• These are questions of function-to address these questions it seemed logical to look at the structure of chromosomes
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Pre-1953-What did we know about chromosomes
• What is significant about 1953?
• Chromosomes made of DNA and protein
• Which of these molecules stored the genetic information?
• Most researchers favored protein. Why?
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History-DNA or protein is the genetic material?
• Griffith-1928
• Avery, McCloud, McCarty-1944
• Hershey and Chase-1952
• Conclusion-DNA was the genetic information in the chromosome
• To understand questions of function regarding genes-we had to know the structure of DNA
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LE 16-2
Living S cells(control)
Living R cells(control)
Heat-killedS cells (control)
Mixture of heat-killedS cells and livingR cells
Mouse dies
Living S cellsare found in blood sample
Mouse healthy Mouse healthy Mouse dies
RESULTS
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LE 16-3
Bacterialcell
Phagehead
Tail
Tail fiber
DNA
100
nm
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Figure 16.2b The Hershey-Chase experiment
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The Race to discover the structure of DNA
• Watson and Crick
• Chargaff
• Pauling
• Wilkins and Franklin
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Figure 16-01
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LE 16-6
Franklin’s X-ray diffractionphotograph of DNA
Rosalind Franklin
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X-ray diffraction insights
1.Double helix with a uniform width of 2nm
2.Purine and pyrimidine bases stacked .34 nm apart
3.Helix makes a turn every 3.4 nm
4.10 layers of nitrogen bases every turn of the helix
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LE 16-UN298
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: widthconsistent with X-ray data
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The Birth of Genetics and Genetic Engineering
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The “Double Helix” paper
• A copy is posted on cell web site-please read it
• Major insights:
• A. DNA is a double helix
• B. The two strands are held together by hydrogen bonding between complementary base pairs (A-T) and G-C)
• DNA is antiparallel
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LE 16-5Sugar–phosphate
backbone
5 end
Nitrogenousbases
Thymine (T)
Adenine (A)
Cytosine (C)
DNA nucleotidePhosphate
3 endGuanine (G)
Sugar (deoxyribose)
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LE 16-8a
Adenine (A) Thymine (T)
Sugar
Sugar
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LE 16-8b
Guanine (G) Cytosine (C)
Sugar
Sugar
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LE 16-7b5 end
3 end
5 end
3 end
Partial chemical structure
Hydrogen bond
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LE 16-7a
Key features of DNA structure
0.34 nm
3.4 nm
1 nm
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LE 16-7c
Space-filling model
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Structure answers a question of function
• Question-How does DNA replicate?
• “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”
• Semi-conservative replication
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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.
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LE 16-9_2
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.
The first step in replication is separation of the two DNA strands.
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LE 16-9_3
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.
The first step in replication is separation of the two DNA strands.
Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.
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Experimental Evidence for Semi-conservative Replication
• Just because something is logical does not mean it is true.
• Three possible mechanisms of DNA replication-
• A. Conservative
• Semi-conservative
• C. Dispersive
• Messelson and Stahl experiment
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LE 16-10
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 moleculeseparate, 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.
Parent cellFirstreplication
Secondreplication
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LE 16-10a
Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix.
Parent cellFirstreplication
Secondreplication
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LE 16-10b
Semiconservative model. The two strands of the parental moleculeseparate, and each functions as a template for synthesis of a new, comple-mentary strand.
Parent cellFirstreplication
Secondreplication
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LE 16-10c
Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA.
Parent cellFirstreplication
Secondreplication
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Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 4)
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DNA replication-It’s more complicated than Watson and Crick thought
• Considerations-DNA replication• 1. DNA must unwind (it’s a double helix)• 2. It’s fast (mammals-50 nucls/sec;
bacteria-500 nucls/sec).• 3.Accuracy-1 mistake/1 billion nucleotides • 4. DNA polymerase limitations-can’t
synthesize denovo; only works in 5’3’ direction
• 5. DNA is antiparallel
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DNA replication proteins
• Several of the replication considerations suggest the involvement of proteins (especially enzymes) in DNA replication
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Consideration #1-DNA must unwind prior to replication
• DNA helicase (unwindase)
• Topoisomerase (relieves twisting)
• Single strand binding proteins
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Consideration #2-Speed of Replication
• Enzymes involved-DNA polymerase (11 forms in eukaryotes)-III is the major replicative enzyme)
• DNA replication is bi-directional
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LE 16-13
New strand
5 end
Phosphate Base
Sugar
Template strand
3 end 5 end 3 end
5 end
3 end
5 end
3 end
Nucleosidetriphosphate
DNA polymerase
Pyrophosphate
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LE 16-12
In eukaryotes, DNA replication begins at may sitesalong the giant DNA molecule of each chromosome.
Two daughter DNA molecules
Parental (template) strand
Daughter (new) strand0.25 µm
Replication fork
Origin of replication
Bubble
In this micrograph, three replicationbubbles are visible along the DNAof a cultured Chinese hamster cell(TEM).
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Consideration #3-Accuracy
• DNA polymerase has “proofreading capabilities”-mismatch repair
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Consideration #4-Limitations of DNA polymerase
• DNA polymerase can’t synthesize a new strand “denovo”-needs a free 3’ OH group to attach the next nucleotide to
• Solution-RNA primase-adds RNA primer (5-10 nucleotides)-later primer removed by a form of DNA polymerase that replaces RNA nucleotides with DNA nucleotides
• Pieces of DNA joined by DNA ligase
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LE 16-15_1
53
Primase joins RNAnucleotides into a primer.
Templatestrand
5 3
Overall direction of replication
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LE 16-15_2
53
Primase joins RNAnucleotides into a primer.
Templatestrand
5 3
Overall direction of replication
RNA primer3
5
35
DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.
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Consideration #4-Limitations of DNA polymerase (continued)
• DNA polymerase only works in 5’3’ direction
• Why is this a problem?• Because of consideration #5-DNA is
antiparallel-One strand runs in the 5’3’ direction; the other runs in the 3’5’ direction
• Solution1- Is there a 3’5’ DNApolymerase? (haven’t found one yet)
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Solution 2-DNA replication occurs differently on the 2 strands
• Leading strand (continuous replication)
• Lagging strand (discontinuous replication)-involvement of Okasaki fragments (approximately 200 nucleotides in length in eukaryotes).
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LE 16-14
Parental DNA
5
3
Leading strand
35
3
5
Okazakifragments
Lagging strand
DNA pol III
Templatestrand
Leading strand
Lagging strand
DNA ligase Templatestrand
Overall direction of replication
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LE 16-15_1
53
Primase joins RNAnucleotides into a primer.
Templatestrand
5 3
Overall direction of replication
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LE 16-15_2
53
Primase joins RNAnucleotides into a primer.
Templatestrand
5 3
Overall direction of replication
RNA primer3
5
35
DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.
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LE 16-15_3
53
Primase joins RNAnucleotides into a primer.
Templatestrand
5 3
Overall direction of replication
RNA primer3
5
35
DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.
Okazakifragment
3
5
5
3
After reaching thenext RNA primer (not
shown), DNA pol IIIfalls off.
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LE 16-15_4
53
Primase joins RNAnucleotides into a primer.
Templatestrand
5 3
Overall direction of replication
RNA primer3
5
35
DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.
Okazakifragment
3
5
5
3
After reaching thenext RNA primer (not
shown), DNA pol IIIfalls off.
33
5
5
After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.
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LE 16-15_5
53
Primase joins RNAnucleotides into a primer.
Templatestrand
5 3
Overall direction of replication
RNA primer3
5
35
DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.
Okazakifragment
3
5
5
3
After reaching thenext RNA primer (not
shown), DNA pol IIIfalls off.
33
5
5
After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.
33
5
5
DNA pol I replaces the RNA with DNA,adding to the 3 endof fragment 2.
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LE 16-15_6
53
Primase joins RNAnucleotides into a primer.
Templatestrand
5 3
Overall direction of replication
RNA primer3
5
35
DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.
Okazakifragment
3
5
5
3
After reaching thenext RNA primer (not
shown), DNA pol IIIfalls off.
33
5
5
After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.
33
5
5
DNA pol I replaces the RNA with DNA,adding to the 3 endof fragment 2.
33
5
5
DNA ligase forms abond between the newestDNA and the adjacent DNAof fragment 1.
The lagging strand in the regionis now complete.
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LE 16-16
5
3Parental DNA
3
5
Overall direction of replication
DNA pol III
Replication fork
Leadingstrand
DNA ligase
Primase
OVERVIEW
PrimerDNA pol III
DNA pol I
Laggingstrand
Laggingstrand
Leadingstrand
Leadingstrand
LaggingstrandOrigin of replication
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Figure 16.15 The main proteins of DNA replication and their functions
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Repair of Damaged DNA
• Environmental factors including UV radiation can damage DNA
• DNA polymerase can repair damage (excision repair)
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LE 16-17
DNA ligase
DNA polymerase
DNA ligase seals thefree end of the new DNAto the old DNA, making thestrand complete.
Repair synthesis bya DNA polymerasefills in the missingnucleotides.
A nuclease enzyme cutsthe damaged DNA strandat two points and the damaged section isremoved.Nuclease
A thymine dimerdistorts the DNA molecule.