The molecular basis of inheritance

The Molecular Basis of
Inheritance

Information: It must contain the information necessary to
make an entire organism
Transmission: It must be passed from parent to offspring
Replication: Able to replicate accurately
Variation: Capable of change to allow evolution
Stable enough to store information for long periods
THE GENETIC MATERIAL

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
© 2011 Pearson Education, Inc.

DNA is the genetic material
• Early in the 20th century, the identification of the
molecules of inheritance loomed as a major
challenge to biologists

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

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
• 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

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 diesMouse healthy Mouse healthy
Living S cells
EXPERIMENT
RESULTS
Figure 16.2

• 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

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

Figure 16.3
Phage
head
Tail
sheath
Tail fiber
DNA
Bacterial
cell
100nm

• In 1952, Alfred Hershey and Martha Chase
performed experiments showing that DNA is the
genetic material of a phage known as T2
• To determine this, 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

Figure 16.4-3
Bacterial cell
Phage
Batch 1:
Radioactive
sulfur
(35
S)
Radioactive
protein
DNA
Batch 2:
Radioactive
phosphorus
(32
P)
Radioactive
DNA
Empty
protein
shell
Phage
DNA
Centrifuge
Centrifuge
Radioactivity
(phage protein)
in liquid
Pellet (bacterial
cells and contents)
Pellet
Radioactivity
(phage DNA)
in pellet
EXPERIMENT

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

The Discovery of RNA as Viral Genetic Material
1. All known cellular organisms have DNA as their
genetic material. Some viruses, however, use RNA
instead.
2. Tobacco mosaic virus (TMV) is composed of RNA
and protein; it contains no DNA. In 1956 Gierer and
Schramm showed that when purified RNA from TMV
is applied directly to tobacco leaves, they develop
mosaic disease. Pretreating the purified RNA with
RNase destroys its ability to cause TMV lesions
3. In 1957 Fraenkel-Conrat and Singer showed that in
TMV infections with viruses containing RNA from
one strain and protein from another, the progeny
viruses were always of the type specified by the
RNA, not by the protein.

Typical tobacco mosaic virus (TMV) particle

Demonstration that RNA is the genetic material in tobacco mosaic virus (TMV)

The Composition and Structure of DNA
and RNA
1. DNA and RNA are polymers composed of
monomers called nucleotides.
2. Each nucleotide has three parts:
a. A pentose (5-carbon) sugar.
b. A nitrogenous base.
c. A phosphate group.
3. The pentose sugar in RNA is ribose, and in DNA
it’s deoxyribose. The only difference is at the 2’
position, where RNA has a hydroxyl (OH) group,
while DNA has only a hydrogen.

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Structures of deoxyribose and ribose in DNA and RNA

The Composition and Structure of DNA
and RNA
4.There are two classes of nitrogenous
bases:
a. Purines (double-ring, nine-membered
structures) include adenine (A) and
guanine (G).
b. Pyrimidines (one-ring, six-membered
structures) include cytosine (C), thymine
(T) in DNA and uracil (U) in RNA.

Structures of the nitrogenous bases in DNA and RNA

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
 Base + sugar  nucleoside
 Example

Adenine + ribose = Adenosine

Adenine + deoxyribose = Deoxyadenosine
 Base + sugar + phosphate(s)  nucleotide
 Example

Adenosine monophosphate (AMP)

Adenosine diphosphate (ADP)

Adenosine triphosphate (ATP)
22

Base always
attached here
Phosphates are
attached here
Adenosine
Adenosine monophosphate
Adenosine diphosphate
Adenine
Phosphate groups
Phosphoester bond
Ribose
H
OP CH2
O–
OO P
O–
O O O
–O P
O–
H
OHHO
O
H
2′3
1′4′
5′
Adenosine triphosphate
NH2
N
H
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
H
N
N
N
23

 Nucleotides are covalently linked together by
phosphodiester bonds
 A phosphate connects the 5’ carbon of one nucleotide
to the 3’ carbon of another
 Therefore the strand has directionality
 5’ to 3’
 In a strand, all sugar molecules are oriented in the
same direction
 The phosphates and sugar molecules form the
backbone of the nucleic acid strand
 The bases project from the backbone
25

Figure 9.10
NH2
N O
N
O
N O
N
Adenine (A)
Guanine (G)
Thymine (T)
BasesBackbone
Cytosine (C)
O
HH
H
H
HH
O
OO
O–
P CH2
O–
HH
H
H
H
HH
O
OO
O
P CH2
O–
NH2
N
N
H
N
N
HH
H
HH
O
OO
O
P CH2
O–
NH2
H
N
N
N
H
N
HH
HOH
HH
O
OO
O
P CH2
O–
Single
nucleotide
Phosphodiester
linkage
Sugar (deoxyribose)
Phosphate
3′
5′
5′
4′ 1′
2′3′
5′
4′ 1′
2′3′
5′
4′ 1′
2′3′
5′
4′ 1′
2′3′
CH3
26

Chapter 2 slide 27
Chemical structures of DNA and RNA

The Discovery of the DNA Double Helix

Erwin Chargaff’s ratios obtained for DNA derived from a
variety of sources showed that the amount of purine always
equals the amount of pyrimidine, and further, that the amount
of G equals C, and the amount of A equals T.
Chargaff’s rules
–The base composition of DNA varies between species
–But, in any species the number of A and T bases are
equal and the number of G and C bases are equal
•The basis for these rules was not understood until
the discovery of the double helix

Building a Structural Model of DNA:
Scientific Inquiry
• After DNA was accepted as the genetic material,
the challenge was to determine how its structure
accounts for its role in heredity
• 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

Figure 16.6
(a) Rosalind Franklin (b) Franklin’s X-ray diffraction
photograph of DNA

• 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

• 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 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)

Figure 16.5
Sugar–phosphate
backbone
Nitrogenous bases
Thymine (T)
Adenine (A)
Cytosine (C)
Guanine (G)
Nitrogenous base
Phosphate
DNA
nucleotide
Sugar
(deoxyribose)
3′ end
5′ end

Figure 16.7
3.4 nm
1 nm
0.34 nm
Hydrogen bond
(a) Key features of
DNA structure
Space-filling
model
(c)(b) Partial chemical structure
3′ end
5′ end
3′ end
5′ end
T
T
A
A
G
G
C
C
C
C
C
C
C
C
C
C
C
G
G
G
G
G
G
G
G
G
T
T
T
T
T
T
A
A
A
A
A
A

• 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

Figure 16.UN01
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: width
consistent with X-ray data

• 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

Sugar
Sugar
Sugar
Sugar
Adenine (A) Thymine (T)
Guanine (G) Cytosine (C)

Main features of Watson and Crick’s three-dimensional model
a. It is two polynucleotide chains
wound around each other in a
right-handed helix.
b. The two chains are antiparallel
c. The sugar-phosphate
backbones are on the outside
of the helix, and the bases
are on the inside, stacked
perpendicularly to the long
axis like the steps of a spiral
staircase.

d. The bases of the two
strands are held together by
hydrogen bonds between
complementary bases (two
for A-T pairs and three for
G-C pairs). Individual H-
bonds are relatively weak
and so the strands can be
separated (by heating, for
example). Complementary
base pairing means that the
sequence of one strand
dictates the sequence of the
other strand.

e. The base pairs are 0.34 nm apart, and one full turn of
the DNA helix takes 3.4 nm, so there are 10 bp in a
complete turn. The diameter of a dsDNA helix is 2 nm.
f. Because of the way the bases H-bond with each other,
the opposite sugar-phosphate backbones are not
equally spaced, resulting in a major and minor groove.
This feature of DNA structure is important for protein
binding.
3. The 1962 Nobel Prize in Physiology or Medicine
was awarded to Francis Crick, James Watson
and Maurice Wilkins (the head of the lab in which
Franklin worked). Franklin had already died, and
so was not eligible.

Different DNA Structures
X ray diffraction studies show that DNA can exist in
different forms
a. A-DNA is the dehydrated form, and so it is not usually found in
cells. It is a right-handed helix with 10.9 bp/turn, with the bases
inclined 13° from the helix axis. A-DNA has a deep and narrow
major groove, and a wide and shallow minor groove.
b. B-DNA is the hydrated form of DNA, the kind normally found in
cells. It is also a right-handed helix, with only 10.0 bp/turn, and
the bases inclined only 2° from the helix axis. B-DNA has a
wide major groove and a narrow minor groove, and its major
and minor grooves are of about the same depth.
c. Z-DNA is a left-handed helix with a zigzag sugar-phosphate
backbone that gives it its name. It has 12.0 bp/turn, with the
bases inclined 8.8° from the helix axis. Z-DNA has a deep
minor groove, and a very shallow major groove. Its existence in
living cells has not been proven.

Space-filling models of different forms of DNA. a) A-DNA b) B-DNA c) Z-DNA

DNA in the Cell
• All known cellular DNA is in the B form.
• A-DNA would not be expected because
it is dehydrated and cells are aqueous.
• Z-DNA has never been found in living
cells, although many organisms have
been shown to contain proteins that will
bind to Z-DNA.

RNA Structure
1. RNA structure is very similar to that of DNA.
a. It is a polymer of ribonucleotides (the sugar is ribose
rather than deoxyribose).
b. Three of its bases are the same (A, G, and C) while it
contains U rather than T.
2. RNA is single-stranded, but internal base pairing
can produce secondary structure in the molecule.
3. Some viruses use RNA for their genomes. In
some it is dsRNA, while in others it is ssRNA.
Double-stranded RNA is structurally very similar
to dsDNA.

 The primary structure of an RNA strand is
much like that of a DNA strand
 RNA strands are typically several hundred to
several thousand nucleotides in length
 In RNA synthesis, only one of the two strands
of DNA is used as a template
RNA Structure
47

Adenine (A)
Guanine
(G)
Uracil (U)
BasesBackbone
Cytosine (C)
O
HH
HH
O
OO
O–
P CH2
O–
HH
HH
O
OO
O
P CH2
O–
NH2
H
N
HH
HH
O
OO
O
P CH2
O–
H
H
HH
OH
HH
O
OO
O
P CH2
O–
Sugar (ribose)
Phosphate
5′
4′ 1′
2′3′
5′
4′ 1′
2′3′
5′
4′ 1′
2′3′
5′
4′ 1′
2′3′
OH
OH
OH
OH
RNA
nucleotide
Phosphodiester
linkage
3′
5′
NH2
OH
H
H
O
NH O
N
N
N
N
N
N
N
N N
N
NH2
H
H
48

 Although usually single-stranded, RNA
molecules can form short double-stranded
regions
 This secondary structure is due to complementary
base-pairing

A to U and C to G
 This allows short regions to form a double helix
 RNA double helices typically
 Are right-handed
 Have the A form with 11 to 12 base pairs per turn
 Different types of RNA secondary structures
are possible
49

A U
A U
U A
G C
C G
C G
A U
U A
U A
C G
C G
C G
C G
C G
A
A
U
U
G
G
C
C
C
(a) Bulge loop (b) Internal loop (c) Multibranched junction (d) Stem-loop
G
C
C
G
U
A
A
U
G
C
G
C
C
G
A
U
A U
A U
G C
A U
U A
G C
C G
C G
G
C
G
C
C
G
A
U
A
U
G
C
C
G
C
G
A
U
A
U
A
U
U
G
G
C
C
C
A U
A U
G C
C G
A
A
A
U
U
U
U
G
G
C
C
Also called
hair-pin
Complementary regions
Non-complementary regions
Held together by
hydrogen bonds
Have bases projecting away
from double stranded regions
50

 Many factors
contribute to the
tertiary structure
of RNA
 For example

Base-pairing and
base stacking
within the RNA
itself

Interactions with
ions, small
molecules and
large proteins
 Figure A: depicts the tertiary structure of tRNAphe
 The transfer RNA that carries phenylalanine
Molecule contains
single- and double-
stranded regions
These spontaneously
fold and interact to
produce this 3-D
structure
Figure A(a) Ribbon model
3 end′
(acceptor
site)5 end′
Double helix
Double helix
Anticodon
51

The Organization of DNA in Chromosomes
• Cellular DNA is organized into chromosomes.
A genome is the chromosome or set of
chromosomes that contains all the DNA of an
organism.
• In prokaryotes the genome is usually a single
circular chromsome. In eukaryotes, the
genome is one complete haploid set of
nuclear chromosomes; mitochondrial and
chloroplast DNA are not included.

Viral Chromosomes
A virus is nucleic acid surrounded by a protein
coat. The nucleic acid may be dsDNA, ssDNA,
dsRNA or ssRNA, and it may be linear or
circular, a single molecule or several segments.
Bacteriophages are viruses that infect bacteria.
Three different types that infect E. coli are good
examples of the variety of chromosome
structure found in viruses.

台大農藝系遺傳學
601 20000
Chapter 2 slide 54
λ chromosome structure varies at stages of lytic infection of E. coli

Prokaryotic Chromosomes
1. The typical prokaryotic genome is one circular
dsDNA chromosome, but some prokaryotes are
more exotic, with a main chromosome and one
or more smaller ones. When a minor
chromosome is dispensable to the life of the cell,
it is called a plasmid. Some examples:
a. Borrelia burgdorferi (Lyme disease in humans) has a
0.91-Mb linear chromosome, plus an additional 0.53-
Mb of DNA in 17 different linear and circular molecules.
b. Agrobacterium tumefaciens (crown gall disease of
plants) has a 3.0-Mb circular chromosome and a 2.1-
Mb linear one.

601 20000
Chapter 2 slide 56
2. Archaebacteria also vary in chromosomal
organization, but only circular forms have been
found. Examples:
a. Methanococcus jannaschii has three chromosomes of
1.66-Mb, 58-kb and 16-kb.
b. Archaeoglobus fulgidus has one 2.2-Mb circular
chromosome.
3. Both Eubacteria and Archaebacteria lack a
membrane-bounded nucleus, hence their
classification as prokaryotes. Their DNA is densely
arranged in a cytoplasmic region called the
nucleoid.
4. In an experiment where E. coli is gently lysed, it
releases one 4.6-Mb circular chromosome, highly
supercoiled (Figures 2.19 and 2.20). A 4.6-Mb
double helix is about 1mm in length, about 103
times longer than an E. coli cell. DNA supercoiling
helps it fit into the cell.

601 20000
Chapter 2 slide 57
Illustration of DNA supercoiling

5. A molecule of B-DNA, with 10bp/turn of the helix, is in relaxed
conformation. If turns of the helix are removed and the molecule
circularized, the DNA will form superhelical turns to compensate for
the added tension.
6. A nick in supercoiled DNA will allow it to return to a relaxed DNA
circle
7. Either overwinding or underwinding DNA will create a structure
where 10bp/turn of the helix is not the most energetically favored
conformation, and supercoils will be induced. Both positive and
negative supercoils will condense the DNA.
8. All organisms contain topoisomerase enzymes to supercoil their
DNA.
9. Prokaryotes also organize their DNA into looped domains, with the
ends of the domains held so that each is supercoiled independently
10. The compaction factor for looped domains is about 10-fold. In E.
coli there are about 100 domains of about 40kb each.

Model for the structure of a bacterial chromosome

Eukaryotic Chromosomes
1. The genome of most prokaryotes consists of
one chromosome, while most eukaryotes
have a diploid number of chromosomes.
2. A genome is the information in one complete
haploid chromosome set. The total amount of
DNA in the haploid genome of a species is its
C value. The structural complexity and the C
value of an organism are not related, creating
the C value paradox.

Chapter 2 slide 61
C value paradox

O
3. The form of eukaryotic chromosomes changes
through the cell cycle:
a. In G1, each chromosome is a single structure
b. In S, chromosomes duplicate into sister chromatids but
remain joined at centromeres through G2
c. At M phase, sister chromatids separate into daughter
chromosomes
4. In G1 eukaryotic chromosomes are linear dsDNA,
and contain about twice as much protein as DNA by
weight. The DNA-protein complex is called
chromatin, and it is highly conserved in all
eukaryotes.

• 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

• 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

Figure 16.22a
DNA double helix
(2 nm in diameter)
DNA, the double helix
Nucleosome
(10 nm in diameter)
Histones
Histones
Histone
tail
H1
Nucleosomes, or “beads on
a string” (10-nm fiber)

Figure 16.22b
30-nm fiber
30-nm fiber
Loops Scaffold
300-nm fiber
Chromatid
(700 nm)
Replicated
chromosome
(1,400 nm)
Looped domains
(300-nm fiber) Metaphase
chromosome

• 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 can undergo chemical modifications that
result in changes in chromatin organization

• Eukaryotic chromosomal DNA molecules have special
nucleotide sequences at their ends called telomeres
• Telomeres are needed for chromosomal replication and
stability. Generally composed of heterochromatin, they
interact with both the nuclear envelope and each other. All
telomeres in a species have the same sequence.
• 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
Telomeres

• 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
• 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

601 20000
Chapter 2 slide 70
Unique-Sequence and Repetitive-Sequence DNA
1. Sequences vary widely in how often they occur
within a genome. The categories are:
a. Unique-sequence DNA, present in one or a few
copies.
b. Moderately repetitive DNA, present in a few to 105
copies.
c. Highly repetitive DNA, present in about 105
–107
copies.
2. Prokaryotes have mostly unique-sequence DNA,
with repeats only of sequences like rRNAs and
tRNAs. Eukaryotes have a mix of unique and
repetitive sequences.
3. Unique-sequence DNA includes most of the genes
that encode proteins, as well as other
chromosomal regions. Human DNA contains about
65% unique sequences.

601 20000
Chapter 2 slide 71
4. Repetitive-sequence DNA includes the moderately and highly
repeated sequences. They may be dispersed throughout the
genome, or clustered in tandem repeats.
5. Dispersed repetitive sequences occur in families that have a
characteristic sequence. Often the same few sequences are
highly repeated, and comprise most of the dispersed repeats in
the genome. Little is known of their function, or indeed whether
they actually serve a function. There are two types of
interspersion patterns found in all eukaryotic organisms:
a. SINEs (short interspersed repeated sequences) with 100–500
bp sequences. An example is the Alu repeats found in some
primates, including humans, where these 200–300 bp repeats
make up 9% of the genome.
b. LINEs (long interspersed repeated sequences) with sequences
of 5 kb or more. The common example in mammals is LINE-1,
with sequences up to 7 kb in length.
6. Tandemly repetitive sequences are common in eukaryotic
genomes, ranging from very short (1–10 bp) sequences to genes
and even longer sequences. This group includes centromere and
telomere sequences, and rRNA and tRNA genes.

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

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

Figure 16.9-3
(a) Parent molecule (b) Separation of
strands
(c)“Daughter” DNA molecules,
each consisting of one
parental strand and one
new 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

• 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)

Figure 16.10
(a) Conservative
model
(b) Semiconservative
model
(c) Dispersive model
Parent
cell
First
replication
Second
replication

• 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
• 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

Figure 16.11
Bacteria
cultured in
medium with
15
N (heavy
isotope)
Bacteria
transferred to
medium with
14
N (lighter
isotope)
DNA sample
centrifuged
after first
replication
DNA sample
centrifuged
after second
replication
Less
dense
More
dense
Predictions: First replication Second replication
Conservative
model
Semiconservative
model
Dispersive
model
21
3 4
EXPERIMENT
RESULTS
CONCLUSION

Getting Started
• Replication begins at particular sites called
origins of replication, where the two DNA
strands are separated, opening up a replication
“bubble”
• A bacterial chromosome has 1 origin of
replication
• 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

Figure 16.12
(a) Origin of replication in an E. coli cell (b) Origins of replication in a eukaryotic cell
Origin of
replication
Parental (template) strand
Double-
stranded
DNA molecule
Daughter (new)
strand
Replication
fork
Replication
bubble
Two daughter
DNA molecules
Origin of replication
Double-stranded
DNA molecule
Parental (template)
strand
Daughter (new)
strand
Bubble Replication fork
Two daughter DNA molecules
0.5µm
0.25µm

• 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 proteins bind to and
stabilize single-stranded DNA
• Topoisomerase corrects “overwinding” ahead of
replication forks by breaking, swiveling, and
rejoining DNA strands

• 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
Topoisomerase
Primase
RNA
primer
Helicase
Single-strand binding
proteins
5′
3′
5′
5′3′
3′

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

• 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, dGTP, dCTP, or dTTP
joins the DNA strand, it loses two phosphate
groups as a molecule of pyrophosphate

Figure 16.14
New strand Template strand
Sugar
Phosphate Base
Nucleoside
triphosphate
DNA
polymerase
Pyrophosphate
5′
5′
5′
5′
3′
3′
3′
3′
OH
OH
OH
P P i
2 P i
P
P
P
A
A
A
A
T 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
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

Figure 16.15
Leading
strand
Lagging
strand
Overview
Origin of replication Lagging
strand
Leading
strand
Primer
Overall directions
of replication
Origin of
replication
RNA primer
Sliding clamp
DNA pol III
Parental DNA
3′
5′
5′
3′
3′
5′
3′
5′
3′
5′
3′
5′

• 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

Figure 16.16a
Origin of replication
Overview
Leading
strand
Leading
strand
Lagging
strand
Lagging strand
Overall directions
of replication
1
2

LE 16-15_6
5′
3′
Primase joins RNA
nucleotides into a primer.
Template
strand
5′ 3′
Overall direction of replication
RNA primer
3′
5′
3′
5′
DNA pol III adds
DNA nucleotides to
the primer, forming
an Okazaki fragment.
Okazaki
fragment
3′
5′
5′
3′
After reaching the
next RNA primer (not
shown), DNA pol III
falls off.
3′
3′
5′
5′
After the second fragment is
primed, DNA pol III adds DNA
nucleotides until it reaches the
first primer and falls off.
3′
3′
5′
5′
DNA pol I replaces
the RNA with DNA,
adding to the 3′ end
of fragment 2.
3′
3′
5′
5′
DNA ligase forms a
bond between the newest
DNA and the adjacent DNA
of fragment 1.
The lagging
strand in the region
is now complete.

Figure 16.17
Overview
Leading
strand
Origin of
replication Lagging
strand
Leading
strandLagging
strand Overall directions
of replicationLeading strand
DNA pol III
DNA pol III Lagging strand
DNA pol I DNA ligase
Primer
Primase
Parental
DNA
5′
5′
5′
5′
5′
3′
3′
3′
3′
3′3 2 1
4

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
• Primase may act as a molecular brake,
coordinating primer placement and the rates of
replication on the leading and lagging strands

Figure 16.18
Parental DNA
DNA pol III
Leading strand
Connecting
protein
Helicase
Lagging strandDNA
pol III
Lagging
strand
template
5′
5′
5′
5′
5′
5′
3′ 3′
3′
3′
3′
3′

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 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

Figure 16.19
Nuclease
DNA
polymerase
DNA
ligase
5′
5′
5′
5′
5′
5′
5′
5′
3′
3′
3′
3′
3′
3′
3′
3′
A thymine dimer
distorts the DNA molecule.
A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed.
Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA ligase seals the
free end of the new DNA
to the old DNA, making the
strand complete.

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

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

LE 16-18
End of parental
DNA strands
5′
3′
Lagging strand 5′
3′
Last fragment
RNA primer
Leading strand
Lagging strand
Previous fragment
Primer removed but
cannot be replaced
with DNA because
no 3′ end available
for DNA polymerase
5′
3′
Removal of primers and
replacement with DNA
where a 3′ end is available
Second round
of replication
5′
3′
5′
3′
Further rounds
of replication
New leading strand
New leading strand
Shorter and shorter
daughter molecules

• 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 the erosion of
genes near the ends of DNA molecules
• It has been proposed that the shortening of
telomeres is connected to aging

The molecular basis of inheritance

The molecular basis of inheritance

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