Biology 201 Chapter 14

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Chapter 14

Lecture and Animation Outline

DNA: The Genetic MaterialChapter 14

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Frederick Griffith – 1928

• Studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia

• 2 strains of Streptococcus– S strain is virulent– R strain is nonvirulent

• Griffith infected mice with these strains hoping to understand the difference between the strains

3

4

• Griffith’s results– Live S strain cells killed the mice– Live R strain cells did not kill the mice– Heat-killed S strain cells did not kill the

mice– Heat-killed S strain + live R strain cells

killed the mice

5

Live NonvirulentStrain of

S. pneumoniae

Mice live

b.

Live VirulentStrain of S. pneumoniae

Mice die

Polysaccharidecoat

a.


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Heat-killed VirulentStrain of S. pneumoniae

Mice live

c.

+

Mixture of Heat-killed Virulentand Live Nonvirulent

Strains of S. pneumoniae

Their lungs contain livepathogenic strain of

S. pneumoniae

Mice die

d.


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• Transformation– Information specifying virulence passed

from the dead S strain cells into the live R strain cells

• Our modern interpretation is that genetic material was actually transferred between the cells

8

Avery, MacLeod, & McCarty – 1944

• Repeated Griffith’s experiment using purified cell extracts

• Removal of all protein from the transforming material did not destroy its ability to transform R strain cells

• DNA-digesting enzymes destroyed all transforming ability

• Supported DNA as the genetic material

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Hershey & Chase –1952

• Investigated bacteriophages– Viruses that infect bacteria

• Bacteriophage was composed of only DNA and protein

• Wanted to determine which of these molecules is the genetic material that is injected into the bacteria

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• Bacteriophage DNA was labeled with radioactive phosphorus (32P)

• Bacteriophage protein was labeled with radioactive sulfur (35S)

• Radioactive molecules were tracked • Only the bacteriophage DNA (as indicated

by the 32P) entered the bacteria and was used to produce more bacteriophage

• Conclusion: DNA is the genetic material

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+

+

Phage grown in radioactive 35S,which is incorporated into phage coat

Virus infectbacteria

Blender separatesphage coat from bacteria

Centrifuge formsbacterial pellet 35S in supernatant

35S-Labeled Bacteriophages

Phage grown in radioactive 32P.which is incorporated into phage DNA

Virus infectbacteria

Blender separatesphage coat from bacteria

Centrifuge formsbacterial pellet 32P in bacteria pellet

32P-Labeled Bacteriophages


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DNA Structure

• DNA is a nucleic acid• Composed of nucleotides

– 5-carbon sugar called deoxyribose– Phosphate group (PO4)

• Attached to 5′ carbon of sugar– Nitrogenous base

• Adenine, thymine, cytosine, guanine– Free hydroxyl group (—OH)

• Attached at the 3′ carbon of sugar

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Purin

esPy

rimid

ines

Adenine Guanine

NH2CC

NN

N

C

HN

C

CH

O

H

H

OC

NC

H

N

C

NH2

H

CH O

O

C

NC

H

N

CH3C

CH

H

O

O

C

NC

H

N

CH

CH

NH2

CC

NN

N

C

HN

C

CHH

Nitrogenous Base

4′

5′

1′

3′ 2′

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7 6

394

51

Phosphate group

Sugar

Nitrogenous base

CH2

N N

O

NNH2

OH in RNA

Cytosine(both DNA and RNA)

Thymine(DNA only)

Uracil(RNA only)

OHH in DNA

O

P

O–

–O O

• Phosphodiester bond– Bond between

adjacent nucleotides– Formed between the

phosphate group of one nucleotide and the 3′ —OH of the next nucleotide

• The chain of nucleotides has a 5′-to-3′ orientation

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BaseCH2

O

5′

3′

O

P

O

OH

CH2

–O O

C

Base

O

PO4

Phosphodiesterbond

Chargaff’s Rules

• Erwin Chargaff determined that– Amount of adenine = amount of thymine– Amount of cytosine = amount of guanine– Always an equal proportion of purines

(A and G) and pyrimidines (C and T)

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Rosalind Franklin

• Performed X-ray diffraction studies to identify the 3-D structure– Discovered that DNA is helical– Using Maurice Wilkins’ DNA

fibers, discovered that the molecule has a diameter of 2 nm and makes a complete turn of the helix every 3.4 nm

a.

b.


Courtesy of Cold Spring Harbor Laboratory Archives

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James Watson and Francis Crick – 1953

• Deduced the structure of DNA using evidence from Chargaff, Franklin, and others

• Did not perform a single experiment themselves related to DNA

• Proposed a double helix structure

Double helix• 2 strands are polymers

of nucleotides• Phosphodiester

backbone – repeating sugar and phosphate units joined by phosphodiester bonds

• Wrap around 1 axis• Antiparallel

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5´

3

O

O

O

O

4

5

1

3 2

4

5

1

3 2

4

5

1

3 2

4

5

1

3 2

5-carbon sugar

Nitrogenous base

Phosphodiester bond

Phosphate group

OH

P

P

P

P


C

C

C

G

G

G

G

G

T

T

T

T

A

A

A

2nm5′ 3′

3.4nm

0.34nm

Minorgroove

Majorgroove

5′3′


Majorgroove

Minorgroove

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• Complementarity of bases

• A forms 2 hydrogen bonds with T

• G forms 3 hydrogen bonds with C

• Gives consistent diameter

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A

H

Sugar

Sugar

Sugar

Sugar

T

G C

N

H

N O

H

CH3

H

HN

N N H NN

N

H

H

H

N O H

H

H N

N HN

N HN N

Hydrogenbond

Hydrogenbond

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DNA Replication

3 possible models1. Conservative model2. Semiconservative model3. Dispersive model

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Conservative

23


Conservative Semiconservative

24


Conservative Semiconservative Dispersive

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Meselson and Stahl – 1958

• Bacterial cells were grown in a heavy isotope of nitrogen, 15N

• All the DNA incorporated 15N • Cells were switched to media containing

lighter 14N• DNA was extracted from the cells at

various time intervals

Meselson and Stahl’s Results

• Conservative model = rejected– 2 densities were not observed after round 1

• Semiconservative model = supported– Consistent with all observations– 1 band after round 1– 2 bands after round 2

• Dispersive model = rejected– 1st round results consistent– 2nd round – did not observe 1 band

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Samples are centrifuged

E. coli

0

1

2

0 rounds 1 round 2 rounds

Bottom

15N medium

14N medium

E. coli cells grownin 15N medium

Cells shifted to14N medium andallowed to grow

DNA

Samples taken atthree time pointsand suspended incesium chloridesolution

Rounds ofreplication

Top

0 min0 rounds

20 min1 round

40 min2 rounds

From M. Meselson and F.W. Stahl/PNAS 44(1958):671


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DNA Replication

• Requires 3 things– Something to copy

• Parental DNA molecule– Something to do the copying

• Enzymes – Building blocks to make copy

• Nucleotide triphosphates

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• DNA replication includes– Initiation – replication begins– Elongation – new strands of DNA are

synthesized by DNA polymerase– Termination – replication is terminated

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P

P

P

P

P

P

P

P

P

P

P

Pyrophosphate

3′

3′

5′

5′

New StrandTemplate Strand

O

HO

OH

O

O

O

O

O

O

O

O

O

OC

C

T

T

T

A

A

A

G

G

A

P

P

P

P

P

PP P

P P

P

P

P

P

3′

3′

5′

5′

New StrandTemplate Strand

O

HO

OH

OH

O

O

O

O

O

O

O

O

OC

C

T

T

A

A

A

G

G

A

Sugar–phosphatebackbone

DNA polymerase III

TO

P


• DNA polymerase– Matches existing DNA bases with

complementary nucleotides and links them– All have several common features

• Add new bases to 3′ end of existing strands• Synthesize in 5′-to-3′ direction• Require a primer of RNA

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5

3

5

5 5 3

3

RNA polymerase makes primer DNA polymerase extends primer

Prokaryotic Replication

• E. coli model• Single circular molecule of DNA• Replication begins at one origin of

replication• Proceeds in both directions around the

chromosome• Replicon – DNA controlled by an origin

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Replisome

ReplisomeTerminationOrigin

Termination

Origin

Origin Origin

Origin

Termination TerminationTermination

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• E. coli has 3 DNA polymerases– DNA polymerase I (pol I)

• Acts on lagging strand to remove primers and replace them with DNA

– DNA polymerase II (pol II)• Involved in DNA repair processes

– DNA polymerase III (pol III)• Main replication enzyme

– All 3 have 3′-to-5′ exonuclease activity – proofreading

– DNA pol I has 5′-to-3′ exonuclase activity

• Unwinding DNA causes torsional strain– Helicases – use energy from ATP to unwind

DNA– Single-strand-binding proteins (SSBs) coat

strands to keep them apart– Topoisomerase prevent supercoiling

• DNA gyrase is used in replication35


Supercoiling

Replisomes

No Supercoiling

Replisomes

DNA gyrase

Semidiscontinous

• DNA polymerase can synthesize only in 1 direction

• Leading strand synthesized continuously from an initial primer

• Lagging strand synthesized discontinuously with multiple priming events– Okazaki fragments

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RNA primer

Open helixand replicate

First RNA primer

Open helix andreplicate further

Lagging strand(discontinuous)

Second RNA primer

Leading strand(continuous)

RNA primer

5′

3′

3′

5′

5′

3′

3′

5′

5′3′

5′

3′

5′

3′


38

• Partial opening of helix forms replication fork

• DNA primase – RNA polymerase that makes RNA primer– RNA will be removed and replaced with

DNA

Leading-strand synthesis– Single priming event– Strand extended by DNA pol III

• Processivity – subunit forms “sliding clamp” to keep it attached

39


a-b: From Biochemistry by Stryer. © 1975, 1981, 1988, 1995 by Lupert Stryer. Used with permission of W.H. Freeman and Company

a. b.

Lagging-strand synthesis– Discontinuous synthesis

• DNA pol III– RNA primer made by primase for each Okazaki fragment– All RNA primers removed and replaced by DNA

• DNA pol I– Backbone sealed

• DNA ligase•Termination occurs at specific site

– DNA gyrase unlinks 2 copies

40

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5′

3′

Primase

RNA primer

Okazaki fragmentmade by DNApolymerase III

Leading strand(continuous)

DNA polymerase I

Lagging strand(discontinuous)

DNA ligase

Replisome

• Enzymes involved in DNA replication form a macromolecular assembly

• 2 main components– Primosome

• Primase, helicase, accessory proteins– Complex of 2 DNA pol III

• One for each strand

42

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Replication forkCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

5 3

New bases β clamp (sliding clamp) Leading strand

Single-strand bindingproteins (SSB)

DNA gyrase

ParentDNA

PrimaseHelicase

3 5

Clamp loader

Open β clamp

Lagging strandOkazaki fragment

5 3

DNA ligase

polymerase IDNA

RNA primer

New bases

polymerase IIIDNA

44

Leading strand

Lagging strand

Primase

Clamp loaderHelicase

DNA polymerase III

DNA gyrase

RNA primer

Single-strandbinding proteins(SSB)

RNA primer

β clamp

1. A DNA polymerase III enzyme is active on each strand. Primase synthesizes new primers for the lagging strand.

5´3´

5´3´

5´3´

RNA primer

Loopgrows

Second Okazakifragment nearscompletion

First Okazakifragment

2. The “loop” in the lagging-strand template allows replication to occur 5´-to- 3´ on both strands, with the complex moving to the left.

5´3´

5´3´

5´3´


5´3´

3. When the polymerase III on the lagging strand hits the previously synthesized fragment, it releases the β clamp and the template strand. DNA polymerase I attaches to remove the primer.

β clampreleases

Laggingstrandreleases

DNA polymerase IIIDNA polymerase I

5´3´

5´3´

45

Clamp loader

4. The clamp loader attaches the β clamp and transfers this to polymerase III, creating a new loop in the lagging-strand template. DNA ligase joins the fragments after DNA polymerase I removes the primers.

DNA ligasepatches “nick”

DNA polymerase Idetaches afterremoving RNA primer

5´3´

5´3´

5´3´

New bases

5. After the β clamp is loaded, the DNA polymerase III on the lagging strand adds bases to the next Okazaki fragment.

Leading strandreplicatescontinuously

Loopgrows

5´3´

5´3´

5´3´


46

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Eukaryotic Replication

• Complicated by– Larger amount of DNA in multiple

chromosomes– Linear structure

• Basic enzymology is similar– Requires new enzymatic activity for

dealing with ends only

48

• Multiple replicons – multiple origins of replications for each chromosome– Not sequence specific; can be adjusted

• Initiation phase of replication requires more factors to assemble both helicase and primase complexes onto the template, then load the polymerase with its sliding clamp unit– Primase includes both DNA and RNA polymerase– Main replication polymerase is a complex of DNA

polymerase epsilon (pol ε) and DNA polymerase delta (pol δ)

Telomeres

• Specialized structures found on the ends of eukaryotic chromosomes

• Protect ends of chromosomes from nucleases and maintain the integrity of linear chromosomes

• Gradual shortening of chromosomes with each round of cell division– Unable to replicate last section of lagging

strand49

50

Leading strand (no problem)Lagging strand (problem at the end)

Last primer

Replication first round

Shortened template

Origin

5´

3´

3´

5´

3´

5´

5´

3´

3´5´

5´

3´

5´

3´

5´

3´

3´

5´

3´

5´

Removed primercannot be replaced

Leadingstrand

Laggingstrand


Primer removal

Replication second round

51

• Telomeres composed of short repeated sequences of DNA

• Telomerase – enzyme makes telomere section of lagging strand using an internal RNA template (not the DNA itself)– Leading strand can be replicated to the end

• Telomerase developmentally regulated– Relationship between senescence and telomere length

• Cancer cells generally show activation of telomerase

52

G

GGGGG

T T

TTT TG

TT

GGG

GGT

TTT

CCCCC AAAA

CCCCC AAAA

Telomere extendedby telomerase

Template RNA ispart of enzyme

Telomerase

Now readyto synthesizenext repeat

5 ́

3 ́

5 ́

3 ́

5 ́

3 ́

Synthesis by telomerase

Telomerase moves andcontinues to extend telomere


53

DNA Repair

• Errors due to replication– DNA polymerases have proofreading ability

• Mutagens – any agent that increases the number of mutations above background level– Radiation and chemicals

• Importance of DNA repair is indicated by the multiplicity of repair systems that have been discovered

54

DNA Repair

Falls into 2 general categories1. Specific repair

– Targets a single kind of lesion in DNA and repairs only that damage

2. Nonspecific– Use a single mechanism to repair multiple

kinds of lesions in DNA

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Photorepair

• Specific repair mechanism• For one particular form of damage caused

by UV light• Thymine dimers

– Covalent link of adjacent thymine bases in DNA• Photolyase

– Absorbs light in visible range– Uses this energy to cleave thymine dimer

56

TA

TA

A A

A A

TA

TA

TT

T T

Thymine dimercleaved

Photolyase

Helix distorted bythymine dimer

Thymine dimer

DNA with adjacent thymines

UV light

Visible light

Photolyase bindsto damaged DNA


Excision repair

• Nonspecific repair• Damaged region is removed and replaced

by DNA synthesis• 3 steps

1. Recognition of damage2. Removal of the damaged region3. Resynthesis using the information on the

undamaged strand as a template

57

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Damaged or incorrect base

Uvr A,B,C complexbinds damaged DNA

DNA polymerase

Excision of damaged strand

Resynthesis by DNA polymerase

Excision repair enzymes recognize damaged DNA

Education

Biology 201 Chapter 14