CAMPBELL BIOLOGY IN FOCUS

Urry • Cain • Wasserman • Minorsky • Jackson • Reece

13
The Molecular
Basis of Inheritance

Lecture Presentations by
Kathleen Fitzpatrick and Nicole Tunbridge

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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 (DNA replication)

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 Figure 13.1

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Concept 13.1: DNA is the genetic material

Concept 13.2: Many proteins work together in

DNA replication and repair

Concept 13.3: A chromosome consists of a DNA

molecule packed together with proteins
Concept 13.4: Understanding DNA structure and
replication makes genetic engineering possible

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Concept 13.1: 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

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 Later work by Oswald Avery and others identified
the transforming substance as DNA.
 Many biologists remained skeptical, mainly because
little was known about DNA and they thought
proteins were better candidates for the genetic
material.

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Evidence That Viral DNA Can Program Cells

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Animation: Hershey-Chase Experiment
Right click slide / Select play
 Figure 13.4
Experiment
Batch 1: Radioactive sulfur (35S) in phage protein
1 Labeled phages 2 Agitation frees outside 3 Centrifuged cells
infect cells. phage parts from cells. form a pellet.
Radioactive 4 Radioactivity
protein (phage protein)
found in liquid

Centrifuge

Pellet
Batch 2: Radioactive phosphorus (32P) in phage DNA
Radioactive
DNA

Centrifuge
4 Radioactivity (phage
Pellet DNA) found 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.

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 Figure 13.5a

Phosphate

3 end

Sugar
DNA (deoxyribose)
nucleotide Nitrogenous
base

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 Figure 13.5

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 Two findings became known as Chargaff’s
rules
 The base composition of DNA varies
between species
 In any species the number of A and T
bases is equal and the number of G and C
bases is equal
 The basis for these rules was not understood
until the discovery of the double helix

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2
-
1
5

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Building a Structural Model of DNA: Scientific
Inquiry
 James Watson and Francis Crick were first to
determine the structure of DNA
 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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 Figure 13.6

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 Figure 13.1

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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 pattern in the photo suggested that the DNA
molecule was made up of two strands, forming a
double helix.

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 Figure 13.7

5 end
C G
C G Hydrogen bond 3 end
G C
G C T A

3.4 nm
T A
G C G C
C G
A T

1 nm C G
T A
C G
G C
C G A T

A T 3 end
A T
0.34 nm
T A 5 end

(a) Key features of (b) Partial chemical structure (c) Space-filling

DNA structure model

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© 2014 Pearson Education, Inc.
 Watson and Crick built models of a double helix to
conform to the X-ray measurements and the
chemistry of DNA
 Franklin had concluded that there were two outer
sugar-phosphate backbones, with the
nitrogenous bases paired in the molecule’s
interior
 Watson built a model in which the backbones were
antiparallel (their subunits run in opposite directions)

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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
data

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 Figure 13.8

Sugar
Sugar
Adenine (A) Thymine (T)

Sugar
Sugar

Guanine (G) Cytosine (C)

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Concept 13.2: 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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 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

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 Figure 13.10
First Second
Parent cell replication replication
(a) Conservative
model

(b) Semiconservative
model

(c) Dispersive
model

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 Figure 13.11
Experiment
1 Bacteria 2 Bacteria
cultured in transferred
medium to medium
with 15N with 14N
(heavy (lighter
isotope) isotope)

Results Less
3 DNA sample 4 DNA sample
centrifuged centrifuged dense
after first after second
replication replication More
dense
Conclusion
Predictions: First replication Second replication

Conservative
model

Semiconservative
model

Dispersive
model

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DNA Replication: A Closer Look

 Replication begins at particular sites called origins

of replication, where the two DNA strands are
separated, opening up a replication “bubble”
 At each end of a bubble is a replication fork, a
Y-shaped region where the parental strands of DNA
are being unwound

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 Figure 13.13

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

cell
Parental
Origin of (template) strand Origin of Double-stranded
replication replication DNA molecule
Daughter
(new) strand
Parental (template) Daughter (new)
Replication strand strand
Double- fork
stranded
DNA Replication
Bubble Replication fork
molecule bubble
Two
daughter DNA
molecules

Two daughter DNA molecules

0.25 m
0.5 m

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 Figure 13.12

Primase

Topoisomerase
3

5 RNA
3 primer
5
Replication
3 fork

Helicase
5

Single-strand binding
proteins

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 Helicases are enzymes that untwist the double helix
at the replication forks
 Single-strand binding proteins bind to and
stabilize single-stranded DNA
 Topoisomerase relieves the strain caused by tight
twisting ahead of the replication fork by breaking,
swiveling, and rejoining DNA strands

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2
0
-
4
0

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Synthesizing a New DNA Strand

 DNA polymerases cannot initiate synthesis of a

polynucleotide; they can only add nucleotides to an
already existing chain base-paired with the template
 The initial nucleotide strand is a short RNA primer
 The enzyme, primase, starts an RNA chain from a
single RNA nucleotide and adds RNA nucleotides
one at a time using the parental DNA as a template
 The primer is short (5–10 nucleotides long)
 The new DNA strand will start from the 3 end of the
RNA primer

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 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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Antiparallel Elongation

 The antiparallel structure of the double helix 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
 Along one template strand of DNA, the DNA
polymerase synthesizes a leading strand
continuously, moving toward the replication fork

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 Figure 13.15a

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 Figure 13.15b
Origin of replication
3
5

5 RNA primer
3
3 Sliding clamp

Parental DNA DNA pol III

5
3
5

5 Continuous elongation
3 in the 5 to 3 direction
3

5
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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
 After formation of Okazaki fragments, DNA
polymerase I removes the RNA primers and
replaces the nucleotides with DNA
 The remaining gaps are joined together by DNA
ligase

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© 2014 Pearson Education, Inc.
 Figure 13.16
Overview
Lagging Origin of replication
Leading strand Lagging
strand strand

Leading RNA primer

strand for fragment 2
Overall directions Okazaki
of replication 5
3 fragment 2 4 DNA pol III
makes Okazaki
3 1 Primase makes
fragment 2.
RNA primer.
5 3
3
Template 5
strand 5 5
5 DNA pol I
3 replaces RNA
RNA primer 2 DNA pol III
3 makes Okazaki with DNA.
for fragment 1
fragment 1.
5 3
3 5
6 DNA ligase forms
5
5 bonds between
3 DNA fragments.
3 DNA pol III
3
detaches.
Okazaki
5
fragment 1 3
5
3
5 Overall direction of replication
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 Figure 13.17b

Leading strand
template
Single-strand
binding proteins

Helicase Leading strand

5 DNA pol III

3 Primer
3 5 Primase
3
Parental DNA
Lagging strand
template

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2
0
-
5
E. coli DNA Polymerases
0

• There are 3 DNA polymerases, the

enzymes that make DNA, found in E. coli:
– pol I
– pol II
– pol III
• E. coli DNA polymerase I was the first
polymerase identified.
• It was discovered in 1958 by Arthur
Kornberg. (1959 Nobel prize)

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Proofreading and Repairing DNA

 DNA polymerases proofread newly made DNA,

replacing any incorrect nucleotides
 In mismatch repair of DNA, other enzymes correct
errors in base pairing
 A hereditary defect in one such enzyme is
associated with a form of colon cancer
 This defect allows cancer-causing errors to
accumulate in DNA faster than normal

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 DNA can be damaged by exposure to harmful
chemical or physical agents such as cigarette
smoke and X-rays; it can also undergo
spontaneous changes
 In nucleotide excision repair, a nuclease cuts
out and replaces damaged stretches of DNA

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 Figure 13.19-3
5 3

3 5

Nuclease

5 3

3 5
DNA
polymerase

5 3

3 5

DNA ligase

5 3

3 5
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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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Replicating the Ends of DNA Molecules

 Limitations of DNA polymerase create problems for

the linear DNA of eukaryotic chromosomes
 The usual replication machinery cannot complete
the 5 ends of daughter strands
 Repeated rounds of replication produce shorter DNA
molecules with uneven ends

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 Eukaryotic chromosomal DNA molecules have special
nucleotide sequences at their ends called telomeres
 Telomeres do not prevent the shortening of DNA
molecules, but they do postpone it
 It has been proposed that the shortening of telomeres
is connected to aging

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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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 Telomerase is not active in most human somatic
cells
 However, it does show inappropriate activity in
some cancer cells
 Telomerase is currently under study as a target for
cancer therapies

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Concept 13.3: A chromosome consists of a DNA
molecule packed together with proteins
 In a bacterium, the DNA is “supercoiled” and found
in a region of the cell called the nucleoid
 Chromatin, a complex of DNA and protein, is found
in the nucleus of eukaryotic cells

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 At interphase, most of the chromatin is compacted
into a 30-nm fiber, which is folded further in some
areas by looping
 Even during interphase, centromeres and some
other parts of chromosomes are highly condensed,
similar to metaphase chromosomes
 This condensed chromatin is called heterochromatin;
the more dispersed, less compacted chromatin is
called euchromatin

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 Dense packing of the heterochromatin makes it
largely inaccessible to the machinery responsible for
transcribing genetic information
 Chromosomes are dynamic in structure; a
condensed region may be loosened or modified as
needed for various cell processes
 For example, histones can undergo chemical
modifications that result in changes in chromatin
organization

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Figure 8-17b

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Table 8-5

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Lysine
Figure 8-18b

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Concept 13.4: Understanding DNA structure and
replication makes genetic engineering possible
 Complementary base pairing of DNA is the basis for
nucleic acid hybridization, the base pairing of one
strand of a nucleic acid to another, complementary
sequence
 Nucleic acid hybridization forms the foundation of
virtually every technique used in genetic
engineering, the direct manipulation of genes for
practical purposes

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 Many bacteria contain plasmids, small circular DNA
molecules that replicate separately from the bacterial
chromosome
 To clone pieces of DNA, researchers first obtain a
plasmid and insert DNA from another source
(“foreign DNA”) into it
 The resulting plasmid is called recombinant DNA

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 Figure 13.22
Bacterium 1 Gene inserted Cell containing gene
into plasmid of interest

Bacterial Plasmid DNA of chromosome

chromosome (“foreign” DNA)
Recombinant Gene of interest
DNA (plasmid)
2 Plasmid put into
bacterial cell

Recombinant
bacterium
3 Host cell grown in culture to form a clone of
cells containing the “cloned” gene of interest
Gene of
interest Protein expressed
from gene of interest
Copies of gene
Protein harvested

4 Basic
research
Gene for pest resistance and various Human growth hormone
inserted into plants applications treats stunted growth

Gene used to alter bacteria Protein dissolves blood clots

for cleaning up toxic waste in heart attack therapy
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 The production of multiple copies of a single gene is
called gene cloning
 Gene cloning is useful to make many copies of a
gene and to produce a protein product
 The ability to amplify many copies of a gene is
crucial for applications involving a single gene

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Using Restriction Enzymes to Make
Recombinant DNA
 Bacterial restriction enzymes cut DNA molecules
at specific DNA sequences called restriction sites
 A restriction enzyme usually makes many cuts,
yielding restriction fragments

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 Figure 13.23-3
Restriction site
5 3
G A AT T C
DNA C T T A AG
3 5
1 Restriction enzyme cuts
the sugar-phosphate
backbones.
3 5
5 3
G
C G
5 3
3 5
Sticky end
5
3
2 DNA fragment added
G
from another molecule 3
cut by same enzyme. 5
Base pairing occurs.

5 3 5 3 5 3
G AAT T C G AAT T C
C T TA A G C T TA A G
3 5 3 5 3 5
3 DNA ligase One possible combination
seals the strands.
5 3

3 5
Recombinant DNA molecule
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 To see the fragments produced by cutting DNA
molecules with restriction enzymes, researchers
use gel electrophoresis
 This technique separates a mixture of nucleic acid
fragments based on length

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 Figure 13.24
Mixture of Power
DNA mol- source
ecules of Cathode Anode
different
sizes
Wells

Gel

(a) Negatively charged DNA molecules will move

toward the positive electrode.

Restriction fragments

(b) Shorter molecules are impeded less than

longer ones, so they move faster through the gel.
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© 2014 Pearson Education, Inc.
Amplifying DNA in Vitro: The Polymerase
Chain Reaction (PCR) and Its Use in Cloning
 The polymerase chain reaction, PCR, can produce
many copies of a specific target segment of DNA
 A three-step cycle brings about a chain reaction that
produces an exponentially growing population of
identical DNA molecules
 The key to PCR is an unusual, heat-stable DNA
polymerase called Taq polymerase.

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 Thomas D. Brock

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 Figure 13.25
Technique 5 3

Target sequence
Genomic DNA 3 5

1 Denaturation 5 3

3 5
2 Annealing

Cycle 1
yields 2 molecules Primers

3 Extension
New
nucleotides

Cycle 2
yields 4 molecules

Cycle 3
yields 8 molecules;
2 molecules
(in white boxes)
match target sequence
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DNA Sequencing

 Once a gene is cloned, complementary base pairing

can be exploited to determine the gene’s complete
nucleotide sequence
 This process is called DNA sequencing

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Concept 13.1: DNA is the genetic material

Concept 13.2: Many proteins work together in

DNA replication and repair

Concept 13.3: A chromosome consists of a DNA

molecule packed together with proteins
Concept 13.4: Understanding DNA structure and
replication makes genetic engineering possible

© 2014 Pearson Education, Inc.