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Chapter 16

The Molecular Basis of Inheritance

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Overview: Life’s Operating Instructions

In 1953, James Watson and Francis Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA
DNA, the substance of inheritance, is the most celebrated molecule of our time
Hereditary information is encoded in DNA and reproduced in all cells of the body
This DNA program directs the development of biochemical, anatomical, physiological, and (to some extent) behavioral traits

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Fig. 16-1

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Concept 16.1: DNA is the genetic material

Early in the 20th century, the identification of the molecules of inheritance loomed as a major challenge to biologists

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The Search for the Genetic Material: Scientific Inquiry

When T. H. Morgan’s group showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material
The key factor in determining the genetic material was choosing appropriate experimental organisms
The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect them

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Evidence That DNA Can Transform Bacteria

The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928
Griffith worked with two strains of a bacterium, one pathogenic and one harmless

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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

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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 dies

Mouse healthy

Living S cells

RESULTS

EXPERIMENT

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In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA
Their conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria
Many biologists remained skeptical, mainly because little was known about DNA

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Evidence That Viral DNA Can Program Cells

More 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

Animation: Phage T2 Reproductive Cycle

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Fig. 16-3

Bacterial

cell

Phage head

Tail sheath

Tail fiber

DNA

100 nm

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In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2
To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection
They concluded that the injected DNA of the phage provides the genetic information

Animation: Hershery-Chase Experiment

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Fig. 16-4-1

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive protein

Radioactive DNA

Batch 1: radioactive sulfur (³⁵S)

Batch 2: radioactive phosphorus (³²P)

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Fig. 16-4-2

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive protein

Radioactive DNA

Batch 1: radioactive sulfur (³⁵S)

Batch 2: radioactive phosphorus (³²P)

Empty protein shell

Phage DNA

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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

Pellet

Pellet (bacterial �cells and contents)

Radioactivity (phage protein) in liquid

Radioactivity (phage DNA) �in pellet

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Additional Evidence That DNA Is the Genetic Material

It was known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group
In 1950, Erwin Chargaff reported that DNA composition varies from one species to the next
This evidence of diversity made DNA a more credible candidate for the genetic material

Animation: DNA and RNA Structure

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Chargaff’s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases

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Fig. 16-5

Sugar–phosphate �backbone �5′ end

Nitrogenous bases

Thymine (T)

Adenine (A)

Cytosine (C)

Guanine (G)

DNA nucleotide

Sugar (deoxyribose) �3′ end

Phosphate

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Building a Structural Model of DNA: Scientific Inquiry

After most biologists became convinced that DNA was the genetic material, the challenge was to determine how its structure accounts for its role
Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure
Franklin produced a picture of the DNA molecule using this technique

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Fig. 16-6

(a) Rosalind Franklin

(b) Franklin’s X-ray diffraction � photograph of DNA

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Fig. 16-6a

(a) Rosalind Franklin

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Fig. 16-6b

(b) Franklin’s X-ray diffraction � photograph of DNA

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Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce that DNA was helical
The X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases
The width suggested that the DNA molecule was made up of two strands, forming a double helix

Animation: DNA Double Helix

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Fig. 16-7

(c) Space-filling model

Hydrogen bond

3′ end

5′ end

3.4 nm

0.34 nm

3′ end

5′ end

(b) Partial chemical structure

(a) Key features of DNA structure

1 nm

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Fig. 16-7a

Hydrogen bond

3′ end

5′ end

3.4 nm

0.34 nm

3′ end

5′ end

(b) Partial chemical structure

(a) Key features of DNA structure

1 nm

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Fig. 16-7b

(c) Space-filling model

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Watson and Crick built models of a double helix to conform to the X-rays and chemistry of DNA
Franklin had concluded that there were two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior

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At first, Watson and Crick thought the bases paired like with like (A with A, and so on), but such pairings did not result in a uniform width
Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray

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Fig. 16-UN1

Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: width consistent with X-ray data

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Watson and Crick reasoned that the pairing was more specific, dictated by the base structures
They determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C)
The Watson-Crick model explains Chargaff’s rules: in any organism the amount of A = T, and the amount of G = C

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Fig. 16-8

Cytosine (C)

Adenine (A)

Thymine (T)

Guanine (G)

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

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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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

Animation: DNA Replication Overview

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Fig. 16-9-1

A

T

G

C

T

A

T

A

G

C

(a) Parent molecule

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Fig. 16-9-2

A

T

G

C

T

A

T

A

G

C

A

T

G

C

T

A

T

A

G

C

(a) Parent molecule

(b) Separation of strands

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Fig. 16-9-3

A

T

G

C

T

A

T

A

G

C

(a) Parent molecule

A

T

G

C

T

A

T

A

G

C

(c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand

(b) Separation of strands

A

T

G

C

T

A

T

A

G

C

A

T

G

C

T

A

T

A

G

C

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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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Fig. 16-10

Parent cell

First replication

Second replication

(a) Conservative model

(b) Semiconserva- tive model

(c) Dispersive model

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Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative model
They labeled the nucleotides of the old strands with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope

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The first replication produced a band of hybrid DNA, eliminating the conservative model
A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model

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Fig. 16-11

EXPERIMENT

RESULTS

CONCLUSION

1

2

4

3

Conservative model

Semiconservative model

Dispersive model

Bacteria cultured in medium containing ¹⁵N

Bacteria transferred to medium containing ¹⁴N

DNA sample centrifuged after 20 min (after first application)

DNA sample centrifuged after 40 min (after second replication)

More dense

Less dense

Second replication

First replication

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Fig. 16-11a

EXPERIMENT

RESULTS

1

3

2

4

Bacteria cultured in medium containing ¹⁵N

Bacteria transferred to medium containing ¹⁴N

DNA sample centrifuged after 20 min (after first application)

DNA sample centrifuged after 20 min (after second replication)

Less dense

More dense

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Fig. 16-11b

CONCLUSION

First replication

Second replication

Conservative model

Semiconservative model

Dispersive model

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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

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Getting Started

Replication begins at special 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

Animation: Origins of Replication

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Fig. 16-12

Origin of replication

Parental (template) strand

Daughter (new) strand

Replication fork

Replication bubble

Two daughter DNA molecules

(a) Origins of replication in E. coli

Origin of replication

Double-stranded DNA molecule

Parental (template) strand

Daughter (new) strand

Bubble

Replication fork

Two daughter DNA molecules

(b) Origins of replication in eukaryotes

0.5 µm

0.25 µm

Double-

stranded

DNA molecule

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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

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Fig. 16-12b

0.25 µm

Origin of replication

Double-stranded DNA molecule

Parental (template) strand

Daughter (new) strand

Bubble

Replication fork

Two daughter DNA molecules

(b) Origins of replication in eukaryotes

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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

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Fig. 16-13

Topoisomerase

Helicase

Primase

Single-strand binding proteins

RNA primer

5′

3′

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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

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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

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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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Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate
dATP supplies adenine to DNA and is similar to the ATP of energy metabolism
The difference is in their sugars: dATP has deoxyribose while ATP has ribose
As each monomer of dATP joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate

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Fig. 16-14

A

C

T

G

C

A

T

New strand 5′ end

Template strand 3′ end

5′ end

3′ end

5′ end

3′ end

Base

Sugar

Phosphate

Nucleoside triphosphate

Pyrophosphate

DNA polymerase

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Antiparallel Elongation

The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication
DNA polymerases add nucleotides only to the free 3′ end of a growing strand; therefore, a new DNA strand can elongate only in the 5′ to 3′ direction

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Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork

Animation: Leading Strand

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Fig. 16-15

Leading strand

Overview

Origin of replication

Lagging strand

Leading strand

Lagging strand

Primer

Overall directions of replication

Origin of replication

RNA primer

“Sliding clamp”

DNA poll III

Parental DNA

5′

3′

5′

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Fig. 16-15a

Overview

Leading strand

Lagging strand

Origin of replication

Primer

Overall directions of replication

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Fig. 16-15b

Origin of replication

RNA primer

“Sliding clamp”

DNA pol III

Parental DNA

3′

5′

3′

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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

Animation: Lagging Strand

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Fig. 16-16

Overview

Origin of replication

Leading strand

Lagging strand

Overall directions of replication

Template strand

RNA primer

Okazaki fragment

Overall direction of replication

1

2

3′

2

1

2

5′

1

3′

5′

3′

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Fig. 16-16a

Overview

Origin of replication

Leading strand

Lagging strand

Overall directions of replication

1

2

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Fig. 16-16b1

Template strand

5′

3′

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Fig. 16-16b2

Template strand

5′

3′

RNA primer

3′

5′

3′

1

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Fig. 16-16b3

Template strand

5′

3′

RNA primer

3′

5′

3′

1

3′

5′

Okazaki fragment

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Fig. 16-16b4

Template strand

5′

3′

RNA primer

3′

5′

3′

1

3′

5′

Okazaki fragment

1

2

3′

5′

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Fig. 16-16b5

Template strand

5′

3′

RNA primer

3′

5′

3′

1

3′

5′

Okazaki fragment

1

2

3′

5′

1

2

3′

5′

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Fig. 16-16b6

Template strand

5′

3′

RNA primer

3′

5′

3′

1

3′

5′

Okazaki fragment

1

2

3′

5′

1

2

3′

5′

1

2

5′

3′

Overall direction of replication

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Table 16-1

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Fig. 16-17

Overview

Origin of replication

Leading strand

Lagging strand

Overall 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′

3′

1

3

2

4

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The DNA Replication Complex

The 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

Animation: DNA Replication Review

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Proofreading and Repairing DNA

DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides
In mismatch repair of DNA, repair enzymes correct errors in base pairing
DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example)
In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA

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Fig. 16-18

Nuclease

DNA polymerase

DNA ligase

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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

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Fig. 16-19

Ends of parental DNA strands

Leading strand

Lagging strand

Last fragment

Previous fragment

Parental strand

RNA primer

Removal of primers and replacement with DNA where a 3′ end is available

Second round of replication

New leading strand

New lagging strand

Further rounds of replication

Shorter and shorter daughter molecules

5′

3′

5′

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Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences called telomeres
Telomeres do not prevent the shortening of DNA molecules, but they do 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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Fig. 16-20

1 µm

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If 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

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The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions
There is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist

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Concept 16.3 A 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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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

Animation: DNA Packing

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Fig. 16-21a

DNA double helix (2 nm in diameter)

Nucleosome

(10 nm in diameter)

Histones

Histone tail

H1

DNA, the double helix

Histones

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

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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

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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

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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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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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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

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Fig. 16-22

RESULTS

Condensin and DNA (yellow)

Outline of nucleus

Condensin (green)

DNA (red at periphery)

Normal cell nucleus

Mutant cell nucleus

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Fig. 16-UN2

Sugar-phosphate backbone

Nitrogenous bases

Hydrogen bond

G

C

A

T

G

A

T

C

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Fig. 16-UN3

DNA pol III synthesizes leading strand continuously

Parental DNA

DNA pol III starts DNA synthesis at 3′ end of primer, continues in 5′ → 3′ direction

Lagging strand synthesized in short Okazaki fragments, later joined by DNA ligase

Primase synthesizes a short RNA primer

5′

3′

5′

3′

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Fig. 16-UN4

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Fig. 16-UN5

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You should now be able to:

Describe the contributions of the following people: Griffith; Avery, McCary, and MacLeod; Hershey and Chase; Chargaff; Watson and Crick; Franklin; Meselson and Stahl
Describe the structure of DNA
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 binding proteins

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Describe the function of telomeres
Compare a bacterial chromosome and a eukaryotic chromosome