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Copyright © 2009 Pearson Education, Inc.

CAMPBELL

BIOLOGYReece • Urry • Cain • Wasserman • Minorsky • Jackson

© 2014 Pearson Education, Inc.

TENTH

EDITION

CAMPBELL

BIOLOGYReece • Urry • Cain • Wasserman • Minorsky • Jackson

© 2014 Pearson Education, Inc.

TENTH

EDITION

16 DNA: The Molecular Basis of Inheritance

Lecture Presentation by

Dr Burns

NVC Biol 120

DNA

Deoxyribonucleic acid – DNA

The blueprint to making proteins!!!

Chromosomes located inside the nucleus

contains long coiled strands of DNA

DNA’s Discovery

Watson and Crick

Rosalind Franklin →

The Players

Crick: Ph.D. student at Cambridge in England working on X-ray Crystallography of the protein hemoglobin

Watson: Young American scientist visiting the lab to do some work on a protein

Both were interested in unraveling the secret of DNA’s structure – it was not what they were supposed to be working on

Wilkins: Working on DNA structure, crystallized

DNA fibers

Franklin: Working at the same university as

Wilkins, just down the hall. Did the X-ray

Crystallography on Wilkins DNA fibers

Linus Pauling: discovered the three dimensional

structure of proteins know as alpha helixes

Chargaff: Discovered that A=T and G=C

Adenine levels always equal thymine levels,

Guanine levels always equal cytosine

2

Franklin gave a talk describing her work with the X-Ray Crystallography, Watson attended but he was not the crystallographer and did not see the implications of her work

Watson and Crick met with Wilkins and he shared Franklin’s work with both of them (without her permission or knowledge)

Watson and Crick put all the pieces of information together.

They built models to help them come up with the structure.

They knew it was a race so they published a one page article in Nature with their ideas – they performed no experiments but were able to see the big picture

Crick, Watson and Wilkins received the Nobel Prize for their work. Rosalind received no credit until much later. She died before the Nobel Prize, the prize is not awarded after a person has died

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Figure 16.7

(a) Key features of

DNA structure

(b) Partial chemical structure

0.34 nm

3′ end

5′ endT

T

T

A

A

A

C

C

C

G

G

G

AT1 nm

TA

C G

CG

AT3.4 nm

CG

CG

C G

C G

3′ end

5′ end

Hydrogen bond

T A

G C

A T

C G

(c) Space-filling

model

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Animation: Hershey-Chase ExperimentRight-click slide / select “Play”

Fig. 14.4

Nucleotide StructureFigure 16.5 Sugar–phosphate

backboneNitrogenous bases

Thymine (T)

Adenine (A)

Cytosine (C)

Guanine (G)

Nitrogenous base

Phosphate

DNA nucleotide

Sugar(deoxyribose)

3 end

5 end

3

DNA Structure

Nucleotides that build DNA have:

One phosphate (ATP has three)

One sugar = deoxyribose

One base.

The nucleotides vary in the type of base – there

are four different bases in DNA: Adenine (A),

Thymine (T), Guanine (G), Cytosine (C)

There is a 5’ end and a 3’ end

Fig. 14.3-1

4

© 2011 Pearson Education, Inc.

Animation: DNA and RNA Structure Right-

click slide / select “Play”

Bonds

The sugars and phosphates link together by covalent bonds to form the rail on the outside = phosphodiester linkage.

The sugars are covalently bound to a base

The complementary bases are attracted to each other by hydrogen bonds

Double Helix

Two strands bonded together by hydrogen

bonds between the bases = weak bonds

Each strand has nucleotides bonded together

covalently by the phosphate and the sugar

Base pairs are two nucleotides, one on each

complementary strand of a DNA molecule

Base Pairs

The bases pair up in a specific manner:

Adenine (A) pairs with Thymine (T)

Guanine (G) pairs with Cytosine (C)

Purines: Adenine and Guanine

Pyrimidines: Thymine and Cytosine

5

Figure 16.8

Sugar

Sugar

Sugar

Sugar

Adenine (A) Thymine (T)

Guanine (G) Cytosine (C)

Remember that on one strand:

The base is covalently bonded to the sugar,

which is covalently bonded to the phosphate

Between the two strands the bases are

bonded together by hydrogen bond

A – T

C – G

The bonds between the sugars and phosphates are

1. Peptide

2. Phosphodiester

3. Hydrogen

4. Ionic

Peptide

Phosphodie

ster

Hydroge

n

Ionic

25% 25%25%25%

The bonds between the bases are

1. Peptide

2. Phosphodiester

3. Hydrogen

4. Ionic

Peptide

Phosphodie

ster

Hydroge

n

Ionic

25% 25%25%25%

Adenine pairs with

1. Thymine

2. Guanine

3. Cytosine

Thym

ine

Guanin

e

Cytosin

e

33%33%33%

Guanine pairs with

1. Thymine

2. Adenine

3. Cytosine

Thym

ine

Adenine

Cytosin

e

33%33%33%

6

The bases are bound to

1. Sugars

2. Phosphates

Suga

rs

Phosphate

s

50%50%

The bases are bound to the sugar by this kind of bond

1. Covalent

2. Phosphodiester

3. Hydrogen

4. Ionic

Covale

nt

Phosphodie

ster

Hydroge

n

Ionic

25% 25%25%25%

The sugar in DNA is

1. Ribose

2. Deoxyribose

3. Glucose

4. Cellulose

Ribose

Deoxyrib

ose

Gluco

se

Cellulo

se

25% 25%25%25%

DNA replication

The relationship between structure and function is

manifest in the double helix

Watson and Crick noted that the specific base

pairing suggested a possible copying mechanism

for genetic material

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DNA Replication

When the structure of DNA was worked

out it became apparent how it happens

It is semiconservative replication

Each strand of DNA is the template for

building new complementary strands

The Basic Principle: Base Pairing to a

Template Strand

Since the two strands of DNA are

complementary, each strand acts as a

template for building a new strand in

replication

In DNA replication, the parent molecule

unwinds, and two new daughter strands are

built based on base-pairing rules

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7

© 2011 Pearson Education, Inc.

Animation: DNA Replication OverviewRight-click slide / select “Play”

Figure 16.9-1

(a) Parent molecule

A

A

A

T

T

T

C

C

G

G

Figure 16.9-2

(a) Parent molecule (b) Separation ofstrands

A

A

A

A

A

A

T

T

T

T

T

T

C

C

C

C

G

G

G

G

Figure 16.9-3

(a) Parent molecule (b) Separation ofstrands

(c) “Daughter” DNA molecules,each consisting of oneparental strand and onenew strand

A

A

A

A

A

A

A

A

A

A

A

A

T

T

T

T

T

T

T

T

T

T

T

T

C

C

C

C

C

C

C

C

G

G

G

G

G

G

G

G

Semiconservative model

Watson and Crick’s semiconservative model

of replication predicts that when a double helix

replicates, each daughter molecule will have

one old strand (derived or “conserved” from the

parent molecule) and one newly made strand

Competing models were the conservative

model (the two parent strands rejoin) and the

dispersive model (each strand is a mix of old

and new)

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8

Figure 16.10

(a) Conservativemodel

(b) Semiconservativemodel

(c) Dispersive model

Parentcell

Firstreplication

Secondreplication

Fig. 14.13

DNA Replication: A Closer Look

The copying of DNA is remarkable in its speed

and accuracy

More than a dozen enzymes and other proteins

participate in DNA replication

© 2011 Pearson Education, Inc.

BioFlix: DNA Replication

Getting Started

Replication begins at particular sites called origins

of replication, where the two DNA strands are

separated, opening up a replication “bubble”

A eukaryotic chromosome may have hundreds or

even thousands of origins of replication

Replication proceeds in both directions from each

origin, until the entire molecule is copied

At the end of each replication bubble is a

replication fork, a Y-shaped region where new

DNA strands are elongating

© 2011 Pearson Education, Inc. © 2011 Pearson Education, Inc.

Animation: Origins of ReplicationRight-click slide / select “Play”

9

Figure 16.12(a) Origin of replication in an E. coli cell (b) Origins of replication in a eukaryotic cell

Origin ofreplication

Parental (template) strand

Double-strandedDNA molecule

Daughter (new)strand

Replicationfork

Replicationbubble

Two daughterDNA molecules

Origin of replication

Double-strandedDNA molecule

Parental (template)strand

Daughter (new)strand

Bubble Replication fork

Two daughter DNA molecules

0.5

m

0.2

5

m

DNA Replication

1. An enzyme, helicase, unwinds the DNA

molecule and breaks the hydrogen bonds

between the base pairs

2. Single-strand binding proteins bind to each

strand and keep them from reforming the double

helix

3. Topoisomerases produce breaks in the DNA

molecule to relieve the stress of unwinding, then

they also repair these breaks.

DNA Replication

Figure 16.13

Topoisomerase

Primase

RNAprimer

Helicase

Single-strand bindingproteins

5

3

5

53

3

DNA Replication

Now the complementary strand needs to be built:

4. Enzymes called DNA polymerases build the new complementary strand by adding new nucleotides to the 3’ end which pair with the old DNA.

5. But DNA polymerase can not start the process. A primer of RNA bases is first built for the complementary strand.

6. An enzyme called primase adds the RNA bases, then DNA polymerase can take over and keep building the complementary strand.

7. The primer is replaced by DNA bases

DNA Replication

8. DNA polymerase builds the new

complementary strand from the 5’ end to the 3’,

by adding the nucleotides to the 3’ end =

leading strand

But the other strand also needs to be replicated

but it can only build new strands by adding to

the 3’ end

DNA Replication DNA Replication

9. The other strand = lagging strand, is build in

short stretches going from 5’ to 3’

10. The short strands being built are called

Okazaki fragments

11. DNA ligase join the Okazaki fragments

DNA Replication

10

Synthesizing a New DNA Strand

Enzymes called DNA polymerases catalyze the

elongation of new DNA at a replication fork

Most DNA polymerases require a primer and a DNA

template strand

The rate of elongation is about 500 nucleotides per

second in bacteria and 50 per second in human

cells

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Energy to power building complementary strand

The incoming nucleotides have three

phosphates, only one is used to bond to the

sugar molecule

The energy needed to build the new DNA

strand comes from taking the other two

phosphates off.

Figure 16.14

New strand Template strand

Sugar

Phosphate Base

Nucleosidetriphosphate

DNApolymerase

Pyrophosphate

5

5

5

5

3

3

3

3

OH

OHP P i

2 P i

A

A

A

A

T T

T

C

C

C

C

C

C

G

G

G

G

Antiparallel Elongation

The antiparallel structure of the double helix

affects replication

DNA polymerases add nucleotides only to the free

3end of a growing strand; therefore, a new DNA

strand can elongate only in the 5to 3direction

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Along one template strand of DNA, the DNA

polymerase synthesizes a leading strand

continuously, moving toward the replication fork

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Antiparallel Elongation

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Animation: Leading Strand Right-

click slide / select “Play”

11

Antiparallel Elongation

To elongate the other new strand, called the

lagging strand, DNA polymerase must work in

the direction away from the replication fork

The lagging strand is synthesized as a series

of segments called Okazaki fragments, which

are joined together by DNA ligase

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Animation: Lagging Strand Right-

click slide / select “Play”

Figure 16.16a

Origin of replication

Overview

Leadingstrand

Leadingstrand

Laggingstrand

Lagging strand

Overall directionsof replication

12

Figure 16.16b-1

Templatestrand

3

35

5

Figure 16.16b-2

Templatestrand

RNA primerfor fragment 1

3

3

3

3

5

5

5

5

1

Figure 16.16b-3

Templatestrand

RNA primerfor fragment 1

Okazakifragment 1

3

3

3

3

3

3

5

5

5

5

5

5

1

1

12

Figure 16.16b-4

Templatestrand

RNA primerfor fragment 1

Okazakifragment 1

RNA primerfor fragment 2

Okazakifragment 2

3

3

3

3

3

3

3

3

5

5

5

5

5

55

5

2

1

1

1

Figure 16.16b-5

Templatestrand

RNA primerfor fragment 1

Okazakifragment 1

RNA primerfor fragment 2

Okazakifragment 2

3

3

3

3

3

3

3

3

3

3

3

5

5

5

5

5

55

55

55

2

2

1

1

1

1

Figure 16.16b-6

Templatestrand

RNA primerfor fragment 1

Okazakifragment 1

RNA primerfor fragment 2

Okazakifragment 2

Overall direction of replication

3

3

3

3

3

3

3

3

3

3

3

3

5

5

5

5

5

55

55

55

5

2

2

21

1

1

1

1

Figure 16.17

Overview

Leadingstrand

Origin of replication Lagging

strand

LeadingstrandLagging

strand Overall directionsof replicationLeading strand

DNA pol III

DNA pol III Lagging strand

DNA pol I DNA ligase

PrimerPrimase

ParentalDNA

5

5

5

5

5

3

3

3

333 2 1

4

Figure 16.17aOverview

Leading

strand

Origin of

replication Lagging

strand

Leading

strandLagging

strand Overall directions

of replication

Leading strand

DNA pol III

PrimerPrimase

ParentalDNA

5

53

3

3

Overview

Leading

strand

Origin of

replicationLagging

strand

Leading

strandLagging

strand Overall directions

of replicationLeading strand

Primer

DNA pol III

DNA pol I

Lagging strand

DNA ligase5

5

5

33

3 3

4

2 1

Figure 16.17b

13

The DNA Replication Complex

The proteins that participate in DNA replication

form a large complex, a “DNA replication

machine”

The DNA replication machine may be stationary

during the replication process

Recent studies support a model in which DNA

polymerase molecules “reel in” parental DNA and

“extrude” newly made daughter DNA molecules

© 2011 Pearson Education, Inc. © 2011 Pearson Education, Inc.

Animation: DNA Replication ReviewRight-click slide / select “Play”

Figure 16.18

Parental DNA

DNA pol III

Leading strand

Connectingprotein

Helicase

Lagging strandDNA pol III

Laggingstrandtemplate

5

5

5

5

5

5

3 3

33

3

3

YouTube - DNA Replication Process

YouTube - DNA Replication (Very realistic

3D animation)

Replicating the Ends of DNA Molecules

Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes

The usual replication machinery provides no way to complete the 5 ends, so repeated rounds of replication produce shorter DNA molecules with uneven ends

This is not a problem for prokaryotes, most of which have circular chromosomes

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Replication at end of DNA

At the end of the DNA strand a small portion

of the strand is not replicated

So we don’t lose important genetic

information, DNA strands have non-coding

end caps

These end caps are called telomeres

14

Figure 16.20

Ends of parentalDNA strands

Leading strand

Lagging strand

Last fragment Next-to-last fragment

Lagging strand RNA primer

Parental strandRemoval of primers andreplacement with DNAwhere a 3 end is available

Second roundof replication

Further roundsof replication

New leading strand

New lagging strand

Shorter and shorter daughter molecules

3

3

3

3

3

5

5

5

5

5Figure 16.20a

Ends of parentalDNA strands

Leading strand

Lagging strand

Last fragment Next-to-last fragment

Lagging strand RNA primer

Parental strandRemoval of primers andreplacement with DNAwhere a 3 end is available

3

3

3

5

5

5

Figure 16.20b

Second roundof replication

Further roundsof replication

New leading strand

New lagging strand

Shorter and shorter daughter molecules

3

3

3

5

5

5

Telomeres

Eukaryotic chromosomal DNA molecules have

special nucleotide sequences at their ends

called telomeres

They postpone the erosion of genes near the

ends of DNA molecules

It has been proposed that the shortening of

telomeres is connected to aging

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Figure 16.21

1 m

Replication at end of DNA

Telomerase build the telomeres.

Embryos have high telomerase activity, as

you age you lose this activity.

Cancer cells have telomerase activity

15

Fig. 14.24-1 Fig. 14.24-2

Mistakes – repair mechanisms

Before a cell can divide, it must make a

complete copy of itself

There are millions of bases that need to be

added to the DNA strands – many chances for

something to go wrong

Enzymes will take out the wrong nucleotide

and replace it with the correct one

Causes of Mutations

Random error – sometimes things just go

wrong.

Mutagens – chemicals that damage the DNA

and cause mutations in replication

Cigarette smoke

Sunlight

Many chemicals (benzene)

Results of Mutations

A few things can happen if DNA mutates

before the cell replicates:

Enzymes can repair the damage

Or – The cell may commit suicide (apoptosis)

Or – The cell may replicate and the mutation

becomes permanent

Proofreading and Repairing DNA

In nucleotide excision repair, an endonuclease cuts

out and DNA polymerase replaces damaged

stretches of DNA

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16

Repair Mechanisms

Photorepair

UV light can cause pyrimidines dimers to occur.

Photolyase uses visible light to break dimer

Nucleotide excision repair

mismatched pairs are recognized and removed

an endonuclease cuts out and DNA

polymerase replaces damaged stretches of

DNA then DNA ligase joins the segements

Fig. 14.25-1

Fig. 14.25-2Figure 16.19

Nuclease

DNA polymerase

DNA ligase

5

5

5

5

5

5

5

5

3

3

3

3

3

3

3

3

Evolutionary Significance of Altered DNA

Nucleotides

Error rate after proofreading repair is low but not

zero

Sequence changes may become permanent and

can be passed on to the next generation

These changes (mutations) are the source of the

genetic variation upon which natural selection

operates

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Which enzyme unwinds the DNA molecule

and breaks the hydrogen bonds between the

base pairs?

1 2 3 4 5

20% 20% 20%20%20%

1. helicase

2. Topoisomerases

3. DNA polymerases

4. Primase

5. DNA ligase

17

Copyright © 2009 Pearson Education, Inc.

Which enzyme produces breaks in the DNA

molecule to relieve the stress of unwinding,

then they also repair these breaks?

1 2 3 4 5

20% 20% 20%20%20%

1. helicase

2. Topoisomerases

3. DNA polymerases

4. Primase

5. DNA ligase

Copyright © 2009 Pearson Education, Inc.

Which enzyme builds the new complementary

strand by adding new nucleotides to the 3’ end

which pair with the old DNA?

1 2 3 4 5

20% 20% 20%20%20%

1. helicase

2. topoisomerases

3. DNA polymerases

4. primase

5. DNA ligase

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Which enzyme adds the RNA bases which starts

the new strands?

1 2 3 4 5

20% 20% 20%20%20%

1. helicase

2. topoisomerases

3. DNA polymerases

4. primase

5. DNA ligase

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Which enzyme joins the Okazaki fragments on the

lagging strand?

1 2 3 4 5

20% 20% 20%20%20%

1. helicase

2. topoisomerases

3. DNA polymerases

4. primase

5. DNA ligase

3’-TAGC-5’ would pair with

1. 3’-ATCG-5’

2. 3’-CGAT-5’

3. 5’-ATCG-3’

4. 5’-CGAT-3’

Chromosome consists of a DNA molecule

packed together with proteins

The bacterial chromosome is a double-stranded,

circular DNA molecule associated with a small

amount of protein

Eukaryotic chromosomes have linear DNA

molecules associated with a large amount of protein

In a bacterium, the DNA is “supercoiled” and found

in a region of the cell called the nucleoid

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18

Chromatin, a complex of DNA and protein,

is found in the nucleus of eukaryotic cells

Chromosomes fit into the nucleus through

an elaborate, multilevel system of packing

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Chromosome consists of a DNA molecule

packed together with proteins

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Animation: DNA PackingRight-click slide / select “Play”

Figure 16.22a

DNA double helix(2 nm in diameter)

DNA, the double helix

Nucleosome(10 nm in diameter)

Histones

Histones

Histonetail

H1

Nucleosomes, or “beads ona string” (10-nm fiber)

Figure 16.22b

30-nm fiber

30-nm fiber

Loops Scaffold

300-nm fiber

Chromatid(700 nm)

Replicatedchromosome(1,400 nm)

Looped domains(300-nm fiber) Metaphase

chromosome

Chromatin undergoes changes in packing during the

cell cycle

At interphase, some chromatin is organized into a

10-nm fiber, but much is compacted into a 30-nm

fiber, through folding and looping

Though interphase chromosomes are not highly

condensed, they still occupy specific restricted

regions in the nucleus

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Chromosome consists of a DNA molecule

packed together with proteins

Figure 16.23

5

m

19

Most chromatin is loosely packed in the nucleus

during interphase and condenses prior to mitosis

Loosely packed chromatin is called euchromatin

During interphase a few regions of chromatin

(centromeres and telomeres) are highly

condensed into heterochromatin

Dense packing of the heterochromatin makes it

difficult for the cell to express genetic information

coded in these regions

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Chromosome consists of a DNA molecule

packed together with proteins

DNA wrapping around proteins

Important Concepts

Know the vocabulary in this lecture

Structure of DNA – and their nucleotides

The four bases, and which are paired together

Be able to recognize the four base structures

Know which bases are purines and Pyrimidines

Type of bonds/linkages

Be able to draw DNA for me (you can use S

and P for sugar and phosphate, ATCG for

bases, 5’ and 3’)

Important Concepts

Be able to describe how is DNA replicated

Semiconservative replication

Steps

Complementary pairing

Direction of building the complementary pair

The role of helicase, Single-strand binding proteins

Topoisomerases, DNA polymerases, DNA ligase

Understand how the leading strand is build vs how

the lagging strand is built, know what Okazaki

fragments are,

Important Concepts

Know what telomers and telomerases are

What supplies the energy to be used to build the

new strand

Be able to identify correctly paired bases and

incorrectly paired bases

Know the repair mechanisms for DNA