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CHAPTER 14LECTURE

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DNA: The Genetic MaterialChapter 14

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

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• 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

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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

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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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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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Pu

rin

esP

yrim

idin

es

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´

2

8

7 6

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4

5

1

O

P

O–

–O

Phosphate group

Sugar

Nitrogenous base

O CH2

N N

O

NNH2

OH in RNA

Cytosine(both DNA and RNA)

Thymine(DNA only)

Uracil(RNA only)

OHH in DNA

• 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

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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´

P

P

P

P

OH

5-carbon sugar

Nitrogenous base

Phosphate group

Phosphodiester bond

O

O

O

O

4´

5´

1´

3´ 2´

4´

5´

1´

3´ 2´

4´

5´

1´

3´ 2´

4´

5´

1´

3´ 2´

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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 N

N

N

H

H

H

N O H

H

H N

N H

N

N HN N

Hydrogenbond

Hydrogenbond

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DNA Replication

• 3 possible models

1. Conservative model

2. Semiconservative model

3. Dispersive model

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Conservative

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Conservative Semiconservative

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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

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• 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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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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• 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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• 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

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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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• 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

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• 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

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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

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Leading strand

Lagging strand

Primase

Clamp loader

Helicase

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´

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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 III

DNA polymerase I

5´3´

5´3´

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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´

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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

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• 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

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• Telomeres composed of short repeated sequences of DNA

• Telomerase – enzyme makes telomere of lagging strand using and 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

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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

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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

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Excision repair

• Nonspecific repair

• Damaged region is removed and replaced by DNA synthesis

• 3 steps1. Recognition of damage

2. Removal of the damaged region

3. Resynthesis using the information on the undamaged strand as a template

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