AP Biology 2006-2007

DNAThe Genetic MaterialGenetics Lecture #5

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Objectives Explain how the structure of DNA was

discovered. Explain how and why DNA replicates

itself.

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Scientific History The march to understanding that DNA is

the genetic material T.H. Morgan (1908) Frederick Griffith (1928) Avery, McCarty & MacLeod (1944) Erwin Chargaff (1947) Hershey & Chase (1952) Watson & Crick (1953) Meselson & Stahl (1958)

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Chromosomes related to phenotype T.H. Morgan

working with Drosophila fruit flies

associated phenotype with specific chromosome white-eyed male had specific

X chromosome

1908 | 1933

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Genes are on chromosomes Morgan’s conclusions

genes are on chromosomes but is it the protein or the

DNA of the chromosomes that are the genes? initially proteins were thought

to be genetic material… Why?

1908 | 1933

What’s so impressiveabout proteins?!

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The “Transforming Principle” 1928

Frederick Griffith Streptococcus pneumonia bacteria

was working to find cure for pneumonia

harmless live bacteria (“rough”) mixed with heat-killed pathogenic bacteria (“smooth”) causes fatal disease in mice

a substance passed from dead bacteria to live bacteria to change their phenotype “Transforming Principle”

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The “Transforming Principle”

Transformation = change in phenotypesomething in heat-killed bacteria could still transmit disease-causing properties

live pathogenicstrain of bacteria

live non-pathogenicstrain of bacteria

mice die mice live

heat-killed pathogenic bacteria

mix heat-killed pathogenic & non-pathogenicbacteria

mice live mice die

A. B. C. D.

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DNA is the “Transforming Principle” Avery, McCarty & MacLeod

purified both DNA & proteins separately from Streptococcus pneumonia bacteria which will transform non-pathogenic bacteria?

injected protein into bacteria no effect

injected DNA into bacteria transformed harmless bacteria into

virulent bacteria

1944

What’s theconclusion?

mice die

AP BiologyOswald Avery Maclyn McCarty Colin MacLeod

Avery, McCarty & MacLeod Conclusion

first experimental evidence that DNA was the genetic material

1944 | ??!!

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Confirmation of DNA Hershey & Chase

classic “blender” experiment worked with bacteriophage

viruses that infect bacteria grew phage viruses in 2 media,

radioactively labeled with either 35S in their proteins 32P in their DNA

infected bacteria with labeled phages

1952 | 1969Hershey

Why useSulfur

vs.Phosphorus?

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Protein coat labeledwith 35S

DNA labeled with 32P

bacteriophages infectbacterial cells

T2 bacteriophagesare labeled with

radioactive isotopesS vs. P

bacterial cells are agitatedto remove viral protein coats

35S radioactivityfound in the medium

32P radioactivity foundin the bacterial cells

Which radioactive marker is found inside the cell?

Which molecule carries viral genetic info?

Hershey & Chase

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Blender experiment Radioactive phage & bacteria in blender

35S phage radioactive proteins stayed in supernatant therefore viral protein did NOT enter bacteria

32P phage radioactive DNA stayed in pellet therefore viral DNA did enter bacteria

Confirmed DNA is “transforming factor”

Taaa-Daaa!

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Hershey & Chase

Alfred HersheyMartha Chase

1952 | 1969Hershey

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Chargaff DNA composition: “Chargaff’s rules”

varies from species to species all 4 bases not in equal quantity bases present in characteristic ratio

humans:

A = 30.9%

T = 29.4%

G = 19.9%

C = 19.8%

1947

That’s interesting!What do you notice?

RulesA = TC = G

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Structure of DNA Watson & Crick

developed double helix model of DNA other leading scientists working on question:

Rosalind FranklinMaurice WilkinsLinus Pauling

1953 | 1962

Franklin Wilkins Pauling

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Watson and CrickWatson Explains Base pairing

CrickWatson

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Rosalind Franklin (1920-1958)

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But how is DNA copied? Replication of DNA

base pairing suggests that it will allow each side to serve as a template for a new strand

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” — Watson & Crick

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Models of DNA Replication Alternative models

become experimental predictions

conservative semiconservative

Can you designa nifty experiment

to verify?

dispersive

1

2

P

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Semiconservative replication Meselson & Stahl

label “parent” nucleotides in DNA strands with heavy nitrogen = 15N

label new nucleotides with lighter isotope = 14N

“The Most Beautiful Experiment in Biology”

1958

parent replicationMake predictions…

15N parent strands

15N/15N

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Predictions

1st round of replication

conservative

15N/15N

14N/14N

semi-conservative

15N/14N

dispersive

15N/14N

conservative

15N/15N

14N/14N

semi-conservative

15N/14N

dispersive

15N/14N

2nd round of replication

14N/14N

15N parent strands

15N/15N

1

2

P

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Franklin Stahl

Matthew Meselson

Matthew Meselson Franklin Stahl

Meselson & Stahl

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Scientific History March to understanding that DNA is the genetic material

T.H. Morgan (1908) genes are on chromosomes

Frederick Griffith (1928) a transforming factor can change phenotype

Avery, McCarty & MacLeod (1944) transforming factor is DNA

Erwin Chargaff (1947) Chargaff rules: A = T, C = G

Hershey & Chase (1952) confirmation that DNA is genetic material

Watson & Crick (1953) determined double helix structure of DNA

Meselson & Stahl (1958) semi-conservative replication

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proteinRNA

The “Central Dogma”

DNAtranscription translation

replication

Flow of genetic information in a cell

AP Biology 2007-2008

DNA Replication

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Double helix structure of DNA

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick

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Directionality of DNA You need to

number the carbons! it matters!

OH

CH2

O

4

5

3 2

1

PO4

N base

ribose

nucleotide

This will beIMPORTANT!!

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The DNA backbone Putting the DNA

backbone together refer to the 3 and 5

ends of the DNA the last trailing carbon

OH

O

3

PO4

base

CH2

O

base

OPO

C

O–O

CH2

1

2

4

5

1

2

3

3

4

5

5

Sounds trivial, but…this will be

IMPORTANT!!

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Anti-parallel strands Nucleotides in DNA

backbone are bonded from phosphate to sugar between 3 & 5 carbons DNA molecule has

“direction” complementary strand runs

in opposite direction

3

5

5

3

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Bonding in DNA

….strong or weak bonds?How do the bonds fit the mechanism for copying DNA?

3

5 3

5

covalentphosphodiester

bonds

hydrogenbonds

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Base pairing in DNA Purines

adenine (A) guanine (G)

Pyrimidines thymine (T) cytosine (C)

Pairing A : T

2 bonds C : G

3 bonds

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Copying DNA Replication of DNA

base pairing allows each strand to serve as a template for a new strand

new strand is 1/2 parent template & 1/2 new DNA semi-conservative

copy process

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DNA Replication Large team of enzymes coordinates replication

Let’s meetthe team…

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Replication: 1st step Unwind DNA

helicase enzyme unwinds part of DNA helix stabilized by single-stranded binding proteins

single-stranded binding proteins replication fork

helicase

I’d love to behelicase & unzip

your genes…

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DNAPolymerase III

Replication: 2nd step

But…We’re missing

something!What?

Where’s theENERGY

for the bonding!

Build daughter DNA strand add new

complementary bases DNA polymerase III

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energy

ATPGTPTTPCTP

Energy of ReplicationWhere does energy for bonding usually come from?

ADPAMPGMPTMPCMPmodified nucleotide

energy

We comewith our own

energy!

And weleave behind a

nucleotide!

Youremember

ATP!Are there other ways

to get energyout of it?

Are thereother energynucleotides?

You bet!

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Energy of Replication The nucleotides arrive as nucleosides

DNA bases with P–P–P P-P-P = energy for bonding

DNA bases arrive with their own energy source for bonding

bonded by enzyme: DNA polymerase III

ATP GTP TTP CTP

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Adding bases can only add

nucleotides to 3 end of a growing DNA strand need a “starter”

nucleotide to bond to

strand only grows 53

DNAPolymerase III

DNAPolymerase III

DNAPolymerase III

DNAPolymerase III

energy

energy

energy

Replication energy

3

3

5B.Y.O. ENERGY!

The energy rulesthe process

5

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Let’s Replicate!!!!DNA Video