Chapter 16

The Molecular Basis of Inheritance

Updated Nov. 2008

Overview: Life’s Operating Instructions

In 1953, James Watson and Francis Crick shook the world With an elegant double-helical model for the

structure of deoxyribonucleic acid, or DNA

Figure 16.1

DNA – Deoxyribonucleic acids

Nucleic acids are unique in their ability to direct their own

replication

It is the DNA program That directs the development of your biochemical,

anatomical, physiological, and (to some extent) behavioral traits

Precise replication of DNA and its transmission to offspring make up heredity

Concept 16.1: DNA is the genetic material

Early in the 20th century After T.H. Morgan’s group showed that genes are

located on chromosomes – proteins and DNA – were the candidates for the genetic material

The identification of the molecules of inheritance loomed as a major challenge to biologists

Until 1940’s, proteins had more support

The Search for the Genetic Material: Scientific Inquiry

Proteins as the genetic material was not consistent with bacteria and viruses

The role of DNA in heredity Was first worked out by studying bacteria and

the viruses that infect them

Evidence That DNA Can Transform Bacteria

Frederick Griffith was studying Streptococcus pneumoniae A bacterium that causes pneumonia in mammals

He worked with two strains of the bacterium A pathogenic strain and a nonpathogenic strain Pathogenic means disease causing

When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic

He called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DNA

Fig. 16-2

Living S cells (control)

Living R cells (control)

Heat-killed S cells (control)

Mixture of heat-killed S cells and living R cells

Mouse diesMouse dies Mouse healthy Mouse healthy

Living S cells

RESULTS

EXPERIMENT

Transformation

Now defined as a change in genotype and phenotype due to the assimilation of external DNA by a cell

Transformation also means conversion of a normal animal cell to a cancerous one (not the same meaning or process)

Evidence That Viral DNA Can Program CellsMore evidence for DNA as the genetic

material came from studies of viruses that infect bacteria

Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research (and other fields like evolutionary biology)

Animation: Phage T2 Reproductive CycleAnimation: Phage T2 Reproductive Cycle

Viruses

Figure 16.3

Phagehead

Tail

Tail fiber

DNA

Bacterialcell

100

nm

Consist of DNA (sometimes RNA) enclosed by a protective coat of protein

Is DNA or Protein the genetic material of phage T2?

Alfred Hershey and Martha Chase Performed experiments showing that DNA is

the genetic material of a phage known as T2

Radioactive sulfur (pink) labeled the proteins (but not DNA)

Radioactive phosphorus (blue) labeled the DNA (but not the proteins)

Fig. 16-4-3

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive protein

Radioactive DNA

Batch 1: radioactive sulfur (35S)

Batch 2: radioactive phosphorus (32P)

Empty protein shell

Phage DNA

Centrifuge

Centrifuge

Pellet

Pellet (bacterial cells and contents)

Radioactivity (phage protein) in liquid

Radioactivity (phage DNA) in pellet

Additional evidence that DNA is the genetic material

Prior to the 1950s, it was already known that DNA

Is a polymer of nucleotides, each consisting of three components: a nitrogenous base, a sugar, and a phosphate group

Sugar-phosphatebackbone

Nitrogenousbases

5 endO–

O P O CH2

5

4O–

HH

O

H

H

H3

1H O

CH3

N

O

NH

Thymine (T)

O

O P O

O–

CH2

HH

O

H

H

H

HN

N

N

H

NH

H

Adenine (A)

O

O P O

O–

CH2

H

HO

H

H

H

H

H H

HN

NN

OCytosine (C)

O

O P O CH2

5

4O–

H

O

H

H3

1

OH

2

H

N

NN H

ON

N HH

H H

Sugar (deoxyribose)3 end

Phosphate

Guanine (G)

DNA nucleotide

2

N

Figure 16.5

Nitrogenous bases

In 1947, Edwin Chargaff developed a set of rules based on a survey of DNA

composition in organisms

Evidence of molecular diversity among species Made DNA a more credible candidate for the

genetic material

Chargaff’s rules

In all organisms, the number of adenines was approximately equal to the number of thymines

The number of guanines was approximately equal to the number of cytosines

The basis for these rules remained unexplained but were key to solving the genetic code

Building a Structural Model of DNA: Scientific Inquiry

Once most biologists were convinced that DNA was the genetic material The challenge was to determine how the

structure of DNA could account for its role in inheritance

Three of the scientists working on this were Linus Pauling (California), Rosalind Franklin (London) and Maurice Wilkins (London)

Maurice Wilkins and Rosalind Franklin

Were using a technique called X-ray crystallography to study molecular structure

Rosalind Franklin Produced a picture of the DNA molecule using

this technique

(a) Rosalind Franklin Franklin’s X-ray diffractionPhotograph of DNA

(b)Figure 16.6 a, b

Rosalind Franklin

Concluded that DNA Was composed of two antiparallel sugar-

phosphate backbones With relatively hydrophobic nitrogenous bases

paired in the molecule’s interior

The nitrogenous bases Are paired in specific combinations: adenine with

thymine, and cytosine with guanine

Watson and Crick deduced that DNA was a double helix

Figure 16.7a, c

C

T

A

A

T

CG

GC

A

C G

AT

AT

A T

TA

C

TA0.34 nm

3.4 nm

(a) Key features of DNA structure

G

1 nm

G

(c) Space-filling model

T

Through observations of the X-ray crystallographic images of DNA

Uniform diameter along the helix

O

–O O

OH

O

–OO

O

H2C

O

–OO

O

H2C

O

–OO

O

OH

O

O

OT A

C

GC

A T

O

O

O

CH2

OO–

OO

CH2

CH2

CH2

5 end

Hydrogen bond3 end

3 end

G

P

P

P

P

O

OH

O–

OO

O

P

P

O–

OO

O

P

O–

OO

O

P

(b) Partial chemical structure

H2C

5 endFigure 16.7b

O

Watson and Crick

Reasoned that there must be additional specificity of pairing

Dictated by the structure of the bases

Each base pair forms a different number of hydrogen bonds

Adenine and thymine form two bonds, cytosine and guanine form three bonds

In April 1953, Watson and Crick published their model in Nature

Hydrogen bondsN H O CH3

N

N

O

N

N

N

N H

Sugar

Sugar

Adenine (A) Thymine (T)

N

N

N

N

Sugar

O H N

H

NH

N OH

H

N

Sugar

Guanine (G) Cytosine (C)Figure 16.8

H

Concept 16.2: Many proteins work together in DNA replication and repair

The relationship between structure and function Is manifest in the double helix

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

(a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

(b) The first step in replication is separation of the two DNA strands.

(c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.

(d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand.

A

C

T

A

G

A

C

T

A

G

A

C

T

A

G

A

C

T

A

G

T

G

A

T

C

T

G

A

T

C

A

C

T

A

G

A

C

T

A

G

T

G

A

T

C

T

G

A

T

C

T

G

A

T

C

T

G

A

T

C

Figure 16.9 a–d

Figure 16.10 a–c

Conservativemodel. The twoparental strandsreassociate after acting astemplates fornew strands,thus restoringthe parentaldouble helix.

Semiconservativemodel. The two strands of the parental moleculeseparate, and each functionsas a templatefor synthesis ofa new, comple-mentary strand.

Dispersivemodel. Eachstrand of bothdaughter mol-ecules containsa mixture ofold and newlysynthesizedDNA.

Parent cellFirstreplication

Secondreplication

DNA replication is semiconservative

Each of the two new daughter molecules will have one old strand, derived from the parent molecule, and one newly made strand

Experiments performed by Meselson and StahlSupported the semiconservative model of

DNA replication

Figure 16.11

Matthew Meselson and Franklin Stahl cultured E. coli bacteria for several generations on a medium containing nucleotide precursors labeled with a heavy isotope of nitrogen, 15N. The bacteria incorporated the heavy nitrogen into their DNA. The scientists then transferred the bacteria to a medium with only 14N, the lighter, more common isotope of nitrogen. Any new DNA that the bacteria synthesized would be lighter than the parental DNA made in the 15N medium. Meselson and Stahl could distinguish DNA of different densities by centrifuging DNA extracted from the bacteria.

EXPERIMENT

The bands in these two centrifuge tubes represent the results of centrifuging two DNA samples from the flask in step 2, one sample taken after 20 minutes and one after 40 minutes.

RESULTS

Bacteriacultured inmediumcontaining15N

Bacteriatransferred tomediumcontaining14N

21

DNA samplecentrifugedafter 20 min(after firstreplication)

3 DNA samplecentrifugedafter 40 min(after secondreplication)

4Lessdense

Moredense

CONCLUSION Meselson and Stahl concluded that DNA replication follows the semiconservative model by comparing their result to the results predicted by each of the three models in Figure 16.10. The first replication in the 14N medium produced a band of hybrid (15N–14N) DNA. This result eliminated the conservative model. A second replication produced both light and hybrid DNA, a result that eliminated the dispersive model and supported the semiconservative model.

First replication Second replication

Conservativemodel

Semiconservativemodel

Dispersivemodel

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

A human cell can copy 6 billion base pairs and divide in only a few hours with only 1 error per 10 billion nucleotides

Getting Started: Origins of ReplicationMuch more is known about replication in

prokaryotes (specifically bacteria) than is known about eukaryotes

But the processes appear to be similarThe replication of a DNA molecule

Begins at special sites called origins of replication, where the two strands are separated

Bidirectional Circular DNA Replication in Bacteria (a prokaryote)

DNA replication occurs in both directions from the origin of replication in the circular DNA found in most bacteria.

In eukaryotes, there may be hundreds or thousands of origin sites per chromosome.

Fig. 16-12a

Origin of replication Parental (template) strand

Daughter (new) strand

Replication fork

Replication bubble

Double-stranded DNA molecule

Two daughter DNA molecules

(a) Origins of replication in E. coli

0.5 µm

Fig. 16-12b

0.25 µm

Origin of replication Double-stranded DNA molecule

Parental (template) strandDaughter (new) strand

Bubble Replication fork

Two daughter DNA molecules

(b) Origins of replication in eukaryotes

Animation: Origins of ReplicationAnimation: Origins of Replication

Proteins in DNA replication

At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating

Helicases are enzymes that untwist the double helix at the replication forks

Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template

Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands

Fig. 16-13

Topoisomerase

Helicase

PrimaseSingle-strand binding proteins

RNA primer

55

5 3

3

3

Initiation

DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end

The initial nucleotide strand is a short RNA primer An enzyme called primase can start an RNA chain

from scratch and adds RNA nucleotides one at a time using the parental DNA as a template

The primer is short (5–10 nucleotides long), and the 3 end serves as the starting point for the new DNA strand

Figure 16.13

New strand Template strand5 end 3 end

Sugar A TBase

C

G

G

C

A

C

T

PP

P

OH

P P

5 end 3 end

5 end 5 end

A T

C

G

G

C

A

C

T

3 endPyrophosphate

2 P

OH

Phosphate

Synthesizing a New DNA Strandat a replication fork is catalyzed by

enzymes called DNA polymerases, which add nucleotides to the 3 end of a growing strand

Nucleosidetriphosphate

DNA Polymerase

The rate of elongation is about 500 nucleotides per second in bacteria, 50 per second in human cells

E. coli has DNA polymerase I and IIIEukaryotes have at least 11 different types

Antiparallel Elongation

How does the antiparallel structure of the double helix affect replication?

(Antiparallel – oriented in opposite directions to each other)

DNA polymerases add nucleotides Only to the free 3end of a growing strand

Directions

3’ end has a free hydroxyl group which is the only place a nucleotide can be added

Along one template strand of DNA, the leading strand DNA polymerase III can synthesize a

complementary strand continuously, moving toward the replication fork

Elongation

To elongate the other new strand of DNA, the lagging strand DNA polymerase III 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 then joined together by DNA ligase

Fig. 16-15

Leading strand

Overview

Origin of replicationLagging strand

Leading strandLagging strand

Primer

Overall directions of replication

Origin of replication

RNA primer

“Sliding clamp”

DNA poll IIIParental DNA

5

3

3

3

3

5

5

5

5

5

Synthesis of DNA

Okazaki fragments are 1,000 – 2,000 nucleotides long in E. coli

They are 100-200 nucleotides long in eukaryotes

DNA ligase joins the fragments together so they form a single DNA strand

Priming DNA Synthesis

DNA polymerases cannot initiate the synthesis of a polynucleotide They can only add nucleotides to the 3 end

The initial nucleotide strand Is an RNA or DNA primer

Primase (and RNA polymerase) joins RNA nucleotides into a primer

Synthesis of the lagging strand

Only one primer is needed for synthesis of the leading strand But for synthesis of the lagging strand, each Okazaki

fragment must be primed separately

DNA pol III adds to the 3’ end of the primer and continues

DNA pol I replaces the RNA nucleotides with DNA versions

DNA ligase forms the bonds between the fragments

Fig. 16-16a

Overview

Origin of replication

Leading strand

Leading strand

Lagging strand

Lagging strand

Overall directions of replication

12

Overall direction of replication

3

3

3

35

35

35

35

3

5

3

5

3

5

3 5

5

1

1

21

12

5

5

12

35

Templatestrand

RNA primer

Okazakifragment

Figure 16.15

Primase joins RNA nucleotides into a primer.

1

DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment.

2

After reaching the next RNA primer (not shown), DNA pol III falls off.

3

After the second fragment is primed. DNA pol III adds DNAnucleotides until it reaches the first primer and falls off.

4

DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2.

5

DNA ligase forms a bond between the newest DNAand the adjacent DNA of fragment 1.

6 The lagging strand in this region is nowcomplete.

7

Table 16-1

Fig. 16-17

OverviewOrigin of replication

Leading strand

Leading strand

Lagging strand

Lagging strandOverall directions

of replication

Leading strand

Lagging strand

Helicase

Parental DNA

DNA pol III

Primer Primase

DNA ligase

DNA pol III

DNA pol I

Single-strand binding protein

5

3

5

5

5

5

3

3

3

313 2

4

The DNA Replication ComplexThe proteins that participate in DNA

replication form a large complex, a “DNA replication machine”

The DNA replication machine is probably 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

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Proofreading and Repairing DNA

Mistakes happen at a rate of 1 per 100,000 base pairs

DNA polymerases proofread newly made DNA Replacing any incorrect nucleotides

In mismatch repair of DNA Repair enzymes correct errors in base pairing

Final error rate is 1 in 10 billion (but 6 billion base pairs in a human chromosome)

Mismatched nucleotides sometimes evade proofreading

In mismatch repair, special enzymes fix incorrectly paired nucleotides

DNA can be damaged by reactive chemicals, radioactive emissions, X-rays and UV light

DNA bases can also undergo spontaneous chemical changes

A hereditary defect in one of these enzymes is responsible for a form of colon cancer

Figure 16.17

Nuclease

DNApolymerase

DNAligase

A thymine dimerdistorts the DNA molecule.1

A nuclease enzyme cutsthe damaged DNA strandat two points and thedamaged section isremoved.

2

Repair synthesis bya DNA polymerasefills in the missingnucleotides.

3

DNA ligase seals theFree end of the new DNATo the old DNA, making thestrand complete.

4

In nucleotide excision repair

Enzymes cut out and replace damaged stretches of DNA

Inherited disorder xeroderma pigmentosum

Having this disorder means hypersensitivity to sunlight

UV light can produce thymine dimers between adjacent thymine nucleotides

Buckles the DNA and interferes with DNA replication

The repair enzyme doesn’t work in this disorder so the mutations are left uncorrected and cause skin cancer

The ends of eukaryotic chromosomal DNA get shorter with each round of replication

Figure 16.18

End of parentalDNA strands

Leading strandLagging strand

Last fragment Previous fragment

RNA primer

Lagging strand

Removal of primers andreplacement with DNAwhere a 3 end is available

Primer removed butcannot be replacedwith DNA becauseno 3 end available

for DNA polymerase

Second roundof replication

New leading strand

New lagging strand 5

Further roundsof replication

Shorter and shorterdaughter molecules

5

3

5

3

5

3

5

3

3

Linear DNA

Prokaryotes don’t have this problem because their DNA is circular

The ends of eukaryotic chromosomal DNA have special nucleotide sequences called telomeres

Telomeres do not contain genes, but multiple repetitions of one short nucleotide sequence

In humans, it is repeated 100 to 1,000 times

Telomeres

postpone the erosion of genes near the ends of DNA molecules

May be connected with aging

Figure 16.19 1 µm

Telomeres and gametes

If the chromosomes of germ cells became shorter in every cell cycle Essential genes would eventually be missing from

the gametes they produce

An enzyme called telomerase Catalyzes the lengthening of telomeres in germ

cells

Telomeres and cancer

Normal shortening of telomeres may protect against cancer by limiting the number of divisions a somatic cell can undergo

Active telomerase has been found in some somatic cells

Telomerase may provide a useful target for cancer diagnosis and chemotherapy

Concept 16.3 A chromosome consists of a DNA molecule packed together with proteinsThe 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

Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells

Histones are proteins that are responsible for the first level of DNA packing in chromatin

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Animation: DNA PackingAnimation: DNA Packing

Fig. 16-21a

DNA double helix (2 nm in diameter)

Nucleosome(10 nm in diameter)

Histones Histone tailH1

DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)

Fig. 16-21b

30-nm fiber

Chromatid (700 nm)

Loops Scaffold

300-nm fiber

Replicated chromosome (1,400 nm)

30-nm fiber Looped domains (300-nm fiber)

Metaphase chromosome

Chromatin is organized into fibers

10-nm fiber DNA winds around histones to form

nucleosome “beads” Nucleosomes are strung together like beads on

a string by linker DNA

30-nm fiber Interactions between nucleosomes cause the

thin fiber to coil or fold into this thicker fiber

300-nm fiber The 30-nm fiber forms looped domains that

attach to proteins

Metaphase chromosome The looped domains coil further The width of a chromatid is 700 nm

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Chromatin

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

Histones

Histones can undergo chemical modifications that result in changes in chromatin organization For example, phosphorylation of a specific

amino acid on a histone tail affects chromosomal behavior during meiosis

You should now be able to:

1. Describe the contributions of the following people: Griffith; Avery, McCary, and MacLeod; Hershey and Chase; Chargaff; Watson and Crick; Franklin; Meselson and Stahl

2. Describe the structure of DNA

3. Describe the process of DNA replication; include the following terms: antiparallel structure, DNA polymerase, leading strand, lagging strand, Okazaki fragments, DNA ligase, primer, primase, helicase, topoisomerase, single-strand

4. Describe the function of telomeres

5. Compare a bacterial chromosome and a eukaryotic chromosome

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings