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DNA and DNA and ReplicationReplication

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History History of DNAof DNA

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History of DNA

• Early scientists thought protein was the cell’s hereditary material because it was more complex than DNA

• Proteins were composed of 20 different amino acids in long polypeptide chains

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TransformationTransformation• Fred Griffith worked with

virulent S and nonvirulent R strain Pneumoccocus bacteria

• He found that R strain could become virulent when it took in DNA from heat-killed S strain

• Study suggested that DNA was probably the genetic material

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Griffith Experiment

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History of DNA• Chromosomes are

made of both DNA and protein

• Experiments on bacteriophage viruses by Hershey & Chase proved that DNA was the cell’s genetic material

Radioactive 32P was injected into bacteria!

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Discovery of DNA Discovery of DNA StructureStructure

• Erwin Chargaff showed the amounts of the four bases on DNA ( A,T,C,G)

• In a body or somatic cell: A = 30.3% T = 30.3% G = 19.5% C = 19.9%

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Chargaff’s RuleChargaff’s Rule• AdenineAdenine must pair with

ThymineThymine• GuanineGuanine must pair with

CytosineCytosine• The bases form weak

hydrogen bonds

G CT A

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DNA StructureDNA Structure•Rosalind Franklin took

diffraction x-ray photographs of DNA crystals

• In the 1950’s, Watson & Crick built the first model of DNA using Franklin’s x-rays

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Rosalind FranklinRosalind Franklin

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DNA DNA StructurStructur

ee

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DNADNA•Two strands coiled called

a double helix•Sides made of a pentose

sugar Deoxyribose bonded to phosphate (PO4) groups by phosphodiester bonds

•Center made of nitrogen bases bonded together by weak hydrogen bonds

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DNA Double HelixDNA Double Helix

NitrogenousNitrogenousBase (A,T,G or C)Base (A,T,G or C)

““Rungs of ladder”Rungs of ladder”

““Legs of ladder”Legs of ladder”

Phosphate &Phosphate &Sugar BackboneSugar Backbone

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HelixHelix

• Most DNA has a Most DNA has a right-right-handhand twist with twist with 10 base 10 base pairspairs in a complete turn in a complete turn

• Left twisted DNA is called Left twisted DNA is called Z-DNAZ-DNA or or southpawsouthpaw DNA DNA

• Hot spotsHot spots occur where occur where right and left twisted right and left twisted DNA meet producingDNA meet producing mutationsmutations

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DNADNA•Stands for

Deoxyribonucleic acid•Made up of subunits

called nucleotidesnucleotides • NucleotideNucleotide made of: made of:

1. Phosphate groupPhosphate group2. 5-carbon sugar5-carbon sugar3. Nitrogenous baseNitrogenous base

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DNA NucleotideDNA Nucleotide

O=P-O O

PhosphatePhosphate GroupGroup

NNitrogenous baseNitrogenous base (A, G, C, or T)(A, G, C, or T)

CH2

O

C1C4

C3 C2

5

SugarSugar(deoxyribose)(deoxyribose)

O

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Pentose SugarPentose Sugar• Carbons are numbered

clockwise 1’ to 5’CH2

O

C1C4

C3 C2

5

SugarSugar(deoxyribose)(deoxyribose)

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DNADNA

P

P

P

O

O

O

1

23

4

5

5

3

3

5

P

P

PO

O

O

1

2 3

4

5

5

3

5

3

G C

T A

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Antiparallel Antiparallel StrandsStrands

• One strand of DNA goes from 5’ to 3’ (sugars)

• The other strand is opposite in direction going 3’ to 5’ (sugars)

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Nitrogenous Nitrogenous BasesBases

• Double ring Double ring PURINESPURINESAdenine (A)Adenine (A)Guanine (G)Guanine (G)

• Single ring Single ring PYRIMIDINESPYRIMIDINES

Thymine (T)Thymine (T)Cytosine (C)Cytosine (C) T or C

A or G

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Base-PairingsBase-Pairings•Purines only pair with

Pyrimidines•Three hydrogen bonds

required to bond Guanine & Cytosine

CG

3 H-bonds

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T A

•Two hydrogen bonds are required to bond Adenine & Thymine

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Question:Question:

•If there is 30% AdenineAdenine, how much CytosineCytosine is present?

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Answer:Answer:•There would be 20%

CytosineCytosine• Adenine (30%) = Adenine (30%) =

Thymine (30%)Thymine (30%)• Guanine (20%) = Guanine (20%) =

Cytosine (20%)Cytosine (20%)• Therefore, Therefore, 60% A-T 60% A-T

and 40% C-Gand 40% C-G

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DNA DNA ReplicatiReplicati

onon

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Replication FactsReplication Facts

• DNA has to be copied DNA has to be copied before a cell dividesbefore a cell divides

• DNA is copied during DNA is copied during the the SS or synthesis phase or synthesis phase of of interphaseinterphase

• New cells will need New cells will need identical identical DNA strandsDNA strands

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Synthesis Phase (S Synthesis Phase (S phase)phase)

• S phase during interphase of the cell cycle

• Nucleus of eukaryotes

Mitosis-prophase-metaphase-anaphase-telophase

G1 G2

Sphase

interphase

DNA replication takesDNA replication takesplace in the S phase.place in the S phase.

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DNA ReplicationDNA Replication

• Begins atBegins at Origins of ReplicationOrigins of Replication• Two strands open forming Two strands open forming

Replication Forks (Y-shaped Replication Forks (Y-shaped region)region)

• New strands grow at the forksNew strands grow at the forks

ReplicationReplicationForkFork

Parental DNA MoleculeParental DNA Molecule

3’

5’

3’

5’

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DNA ReplicationDNA Replication• As the 2 DNA strands open at As the 2 DNA strands open at

the origin, the origin, Replication Replication BubblesBubbles form form

• Eukaryotic chromosomes have MANY bubbles

• Prokaryotes (bacteria) have a single bubble

Bubbles Bubbles

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DNA ReplicationDNA Replication

• Enzyme Enzyme HelicaseHelicase unwinds and separates unwinds and separates the 2 DNA strands by the 2 DNA strands by breaking the breaking the weak weak hydrogen bondshydrogen bonds

• Single-Strand Binding Single-Strand Binding ProteinsProteins attach and keep the 2 DNA strands separated and untwisted

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DNA ReplicationDNA Replication• Enzyme Enzyme TopoisomeraseTopoisomerase

attaches to the 2 forks of the bubble to relieve stressrelieve stress on the DNA moleculeDNA molecule as it separatesEnzyme

DNA

Enzyme

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DNA ReplicationDNA Replication• BeforeBefore new DNA strands can

form, there must be RNA RNA primersprimers present to start the addition of new nucleotides

• PrimasePrimase is the enzyme that synthesizes the RNA Primer

• DNA polymerase can then add the new nucleotides

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DNA ReplicationDNA Replication• DNA polymeraseDNA polymerase can only add can only add

nucleotides to the nucleotides to the 3’ end3’ end of of the DNA the DNA

• This causes the This causes the NEWNEW strand to strand to be built in a be built in a 5’ to 3’ direction5’ to 3’ direction

RNARNAPrimerPrimerDNA PolymeraseDNA Polymerase

NucleotideNucleotide

5’

5’ 3’

Direction of ReplicationDirection of Replication

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Remember HOW the Remember HOW the Carbons Are Numbered!Carbons Are Numbered!

OO=P-O O

PhosphatePhosphate GroupGroup

NNitrogenous baseNitrogenous base (A, G, C, or T)(A, G, C, or T)

CH2

O

C1C4

C3 C2

5

SugarSugar(deoxyribose)(deoxyribose)

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Remember the Strands are Remember the Strands are AntiparallelAntiparallel

P

P

P

O

O

O

1

23

4

5

5

3

3

5

P

P

PO

O

O

1

2 3

4

5

5

3

5

3

G C

T A

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Synthesis of the New Synthesis of the New DNA StrandsDNA Strands

• The The Leading StrandLeading Strand is synthesized as a single single strand strand from the point of origin toward the opening replication fork

RNARNAPrimerPrimerDNA PolymeraseDNA PolymeraseNucleotidesNucleotides

3’5’

5’

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Synthesis of the New DNA Synthesis of the New DNA StrandsStrands

• The The Lagging StrandLagging Strand is is synthesized discontinuouslydiscontinuously against overall direction of replication

• This strand is made in MANY short segments It is replicated from the replication fork toward the origin

RNA PrimerRNA Primer

Leading StrandLeading Strand

DNA PolymeraseDNA Polymerase

5’

5’

3’

3’

Lagging StrandLagging Strand

5’

5’

3’

3’

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Lagging Strand Lagging Strand SegmentsSegments

• Okazaki FragmentsOkazaki Fragments - - series of short segments on the lagging strandlagging strand

• Must be joined together by Must be joined together by an an enzymeenzyme

Lagging Strand

RNARNAPrimerPrimer

DNADNAPolymerasePolymerase

3’

3’

5’

5’

Okazaki FragmentOkazaki Fragment

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Joining of Okazaki Joining of Okazaki FragmentsFragments

• The enzyme The enzyme LigaseLigase joins the joins the Okazaki fragments together Okazaki fragments together to make one strandto make one strand

Lagging Strand

Okazaki Fragment 2Okazaki Fragment 2

DNA ligaseDNA ligase

Okazaki Fragment 1Okazaki Fragment 1

5’

5’

3’

3’

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Replication of Replication of StrandsStrands

Replication Fork

Point of Origin

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Proofreading New Proofreading New DNADNA

• DNA polymerase initially DNA polymerase initially makes about makes about 1 in 10,0001 in 10,000 base base pairing errorspairing errors

• EnzymesEnzymes proofread and proofread and correct these mistakescorrect these mistakes

• The new error rate for DNA The new error rate for DNA that has been proofread is that has been proofread is 1 1 in 1 billionin 1 billion base pairing errors base pairing errors

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Semiconservative Model Semiconservative Model of Replicationof Replication

• Idea presented by Idea presented by Watson & CrickWatson & Crick• TheThe two strands of the parental

molecule separate, and each acts as a template for a new complementary strand

• New DNA consists of 1 PARENTAL (original) and 1 NEW strand of DNA

Parental DNA

DNA Template

New DNA

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DNA Damage & DNA Damage & RepairRepair

• Chemicals & ultraviolet radiation damage the DNA in our body cells

• Cells must continuously repair DAMAGED DNA

• Excision repair occurs when any of over 50 repair enzymes remove damaged parts of DNA

• DNA polymerase and DNA ligase replace and bond the new nucleotides together

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Question:Question:

•What would be the complementary DNA strand for the following DNA sequence?

DNA 5’-CGTATG-3’DNA 5’-CGTATG-3’

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Answer:Answer:

DNA 5’-GCGTATG-3’DNA 5’-GCGTATG-3’

DNA 3’-CGCATAC-5’DNA 3’-CGCATAC-5’

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