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DNA andReplication
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Historyof DNA
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History of DNA• Early scientists thoughtprotein was the cell’shereditary material becauseit was more complex thanDNA
• Proteins were composed of20 different amino acids inlong polypeptide chains
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Transformation• Fred Griffith worked withvirulent S and nonvirulent Rstrain Pneumoccocus bacteria
• He found that R strain couldbecome virulent when it took inDNA from heat-killed S strain
• Study suggested that DNA wasprobably the genetic material
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Griffith Experiment
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History of DNA• Chromosomes are madeof both DNA andprotein
• Experiments onbacteriophage virusesby Hershey & Chaseproved that DNA wasthe cell’s geneticmaterial
Radioactive 32P was injected into bacteria!copyright cmassengale
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Discovery of DNAStructure
• Erwin Chargaff showed theamounts of the four bases onDNA ( 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 Rule• Adenine must pair withThymine
• Guanine must pair withCytosine
• The bases form weakhydrogen bonds
G CT Acopyright cmassengale
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DNA Structure• Rosalind Franklin tookdiffraction x-rayphotographs of DNAcrystals
• In the 1950’s, Watson &Crick built the first modelof DNA using Franklin’sx-rays
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Rosalind Franklin
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DNAStructure
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DNA• Two strands coiled calleda double helix
• Sides made of a pentosesugar Deoxyribose bondedto phosphate (PO4) groupsby phosphodiester bonds
• Center made of nitrogenbases bonded together byweak hydrogen bonds
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DNA Double Helix
NitrogenousBase (A,T,G or C)
“Rungs of ladder”
“Legs of ladder”
Phosphate &Sugar Backbone
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Helix• Most DNA has a right-handtwist with 10 base pairs in acomplete turn
• Left twisted DNA is calledZ-DNA or southpaw DNA
• Hot spots occur where rightand left twisted DNA meetproducing mutations
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DNA• Stands forDeoxyribonucleic acid
• Made up of subunitscalled nucleotides
• Nucleotide made of:1. Phosphate group2. 5-carbon sugar3. Nitrogenous base
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DNA Nucleotide
O=P-OO
PhosphateGroup
NNitrogenous base(A, G, C, or T)
CH2
O
C1C4
C3 C2
5
Sugar(deoxyribose)
O
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Pentose Sugar• Carbons are numbered clockwise1’ to 5’
CH2
O
C1C4
C3 C2
5
Sugar(deoxyribose)
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DNA
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 Strands• One strand of
DNA goes from5’ to 3’ (sugars)
• The otherstrand isopposite indirection going3’ to 5’ (sugars)
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Nitrogenous Bases• Double ring PURINES
Adenine (A)Guanine (G)
• Single ring PYRIMIDINESThymine (T)Cytosine (C) T or C
A or G
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Base-Pairings• Purines only pair withPyrimidines
• Three hydrogen bondsrequired to bond Guanine& Cytosine
CG
3 H-bonds
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T A
•Two hydrogen bonds arerequired to bond Adenine &Thymine
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Question:
• If there is 30%Adenine, how muchCytosine is present?
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Answer:• There would be 20%Cytosine
• Adenine (30%) = Thymine(30%)
• Guanine (20%) = Cytosine(20%)
• Therefore, 60% A-T and40% C-G
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DNAReplication
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Replication Facts• DNA has to be copiedbefore a cell divides
• DNA is copied during the Sor synthesis phase ofinterphase
• New cells will need identicalDNA strands
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Synthesis Phase (S phase)• S phase during interphase of the
cell cycle• Nucleus of eukaryotes
Mitosis-prophase-metaphase-anaphase-telophase
G1 G2
Sphase
interphase
DNA replication takesplace in the S phase.
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DNA Replication• Begins at Origins of Replication• Two strands open forming Replication
Forks (Y-shaped region)• New strands grow at the forks
ReplicationFork
Parental DNA Molecule
3’
5’
3’
5’copyright cmassengale
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DNA Replication• As the 2 DNA strands open at
the origin, Replication Bubblesform
• Prokaryotes (bacteria) have asingle bubble
• Eukaryotic chromosomes haveMANY bubbles
Bubbles Bubbles
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DNA Replication• Enzyme Helicase unwindsand separates the 2 DNAstrands by breaking theweak hydrogen bonds
• Single-Strand BindingProteins attach and keepthe 2 DNA strandsseparated and untwisted
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DNA Replication• Enzyme Topoisomerase attachesto the 2 forks of the bubble torelieve stress on the DNAmolecule as it separatesEnzyme
DNA
Enzyme
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DNA Replication• Before new DNA strands can
form, there must be RNAprimers present to start theaddition of new nucleotides
• Primase is the enzyme thatsynthesizes the RNA Primer
• DNA polymerase can then addthe new nucleotides
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DNA Replication• DNA polymerase can only addnucleotides to the 3’ end of theDNA
• This causes the NEW strand to bebuilt in a 5’ to 3’ direction
RNAPrimerDNA Polymerase
Nucleotide
5’
5’ 3’
Direction of Replicationcopyright cmassengale
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Remember HOW theCarbons Are Numbered!
OO=P-O
O
PhosphateGroup
NNitrogenous base
(A, G, C, or T)
CH2
O
C1C4
C3 C2
5
Sugar(deoxyribose)
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Remember the Strands areAntiparallel
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 DNAStrands
• The Leading Strand issynthesized as a single strandfrom the point of origin towardthe opening replication fork
RNAPrimerDNA PolymeraseNucleotides
3’5’
5’
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Synthesis of the New DNAStrands
• The Lagging Strand is synthesizeddiscontinuously against overall direction ofreplication
• This strand is made in MANY short segmentsIt is replicated from the replication forktoward the origin
RNA Primer
Leading Strand
DNA Polymerase
5’
5’
3’
3’
Lagging Strand
5’
5’
3’
3’ copyright cmassengale
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Lagging Strand Segments• Okazaki Fragments - series of
short segments on the laggingstrand
• Must be joined together by anenzyme
Lagging Strand
RNAPrimer
DNAPolymerase
3’
3’
5’
5’
Okazaki Fragment
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Joining of Okazaki Fragments
• The enzyme Ligase joins theOkazaki fragments together tomake one strand
Lagging Strand
Okazaki Fragment 2
DNA ligase
Okazaki Fragment 1
5’
5’
3’
3’
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Replication of StrandsReplicationFork
Point of Origin
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Proofreading New DNA
• DNA polymerase initially makesabout 1 in 10,000 base pairingerrors
• Enzymes proofread and correctthese mistakes
• The new error rate for DNA thathas been proofread is 1 in 1 billionbase pairing errors
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Semiconservative Model ofReplication
• Idea presented by Watson & Crick• The two strands of the parental
molecule separate, and each acts as atemplate for a new complementarystrand
• New DNA consists of 1PARENTAL (original) and 1 NEWstrand of DNA
Parental DNA
DNA Template
New DNA
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DNA Damage & Repair• 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 removedamaged parts of DNA
• DNA polymerase and DNA ligasereplace and bond the new nucleotidestogether
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Question:
• What would be thecomplementary DNAstrand for the followingDNA sequence?
DNA 5’-CGTATG-3’
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Answer:
DNA 5’-CGTATG-3’DNA 3’-GCATAC-5’
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