DNA Structure 13

The Deoxyribonucleotides 13
The DNA Chain 14
The 5′ and 3′ Ends 14
Base Pairing 16
Antiparallel Construction 17
The Major and Minor Grooves 17
The Mechanism of DNA
Replication 17
Deoxyribonucleotide Precursor
Synthesis 17
Replication of the Bacterial
Chromosome 19
CHAPTER 1
Replication of Double-Stranded DNA 23
Replication Errors 26
Editing 26
RNA Primers and Editing 27 The Bacterial Chromosome:
Impediments to DNA Replication 28
Damaged DNA and DNA Polymerase III 28
Mechanisms To Deal with Impediments on
DNA Structure, Replication,
Template DNA Strands 28
Physical Blocks to Replication Forks 30 and Segregation
Replication of the Bacterial
Chromosome and Cell Division 31
Structure of the Bacterial Chromosome 31
Replication of the Bacterial
Chromosome 31
Initiation of Chromosome Replication 32
RNA Priming of Initiation 33 DNA Structure
Termination of Chromosome

Replication 33
Chromosome Segregation 34
Coordination of Cell Division with
Replication of the Chromosome 43
Timing of Initiation of Replication 45
The Bacterial Nucleoid 47
Supercoiling in the Nucleoid 47
Topoisomerases 49
The Bacterial Genome 50
Antibiotics That Affect Replication and
DNA Structure 51
Antibiotics That Block Precursor
Synthesis 51
Antibiotics That Block Polymerization of
Deoxynucleotides 52
Antibiotics That Affect DNA Structure 52
Antibiotics That Affect Gyrase 52
Molecular Biology Manipulations
with DNA 53
Restriction Endonucleases 53
Hybridizations 56
Applications of the Enzymes Used in
DNA Replication 58
Polymerase Chain Reaction 58
BOX 1.1 Structural Features of
Bacterial Genomes 37
BOX 1.2 Advanced Genome-Sequencing
Techniques 59
T
HE SCIENCE OF MOLECULAR GENETICS began with the determination
of the structure of DNA. Experiments with bacteria and phages (i.e.,
viruses that infect bacteria) in the late 1940s and early 1950s, as well
as the presence of DNA in chromosomes of higher organisms, had impli-
cated this macromolecule as the hereditary material (see the introduction).
In the 1930s, biochemical studies of the base composition of DNA by Erwin
Chargaff established that the amount of guanine always equals the amount
of cytosine and that the amount of adenine always equals the amount of
thymine, independent of the total base composition of the DNA. In the early
1950s, X-ray diffraction studies by Rosalind Franklin and Maurice Wilkins
showed that DNA is a double helix. Finally, in 1953, Francis Crick and
James Watson put together the chemical and X-ray diffraction information
in their famous model of the structure of DNA. This story is one of the most
dramatic in the history of science and has been the subject of many historical
treatments, some of which are listed at the end of this chapter.
Figure 1.1 illustrates the Watson-Crick structure of DNA, in which two
strands wrap around each other to form a double helix. These strands can
be extremely long, even in a simple bacterium, extending up to 1 mm—a
thousand times longer than the bacterium itself. In a human cell, the strands
that make up a single chromosome (which is one DNA molecule) are hun-
dreds of millimeters, or many inches, long.
The Deoxyribonucleotides
If we think of DNA strands as chains, deoxyribonucleotides form the
links. Figure 1.2 shows the basic structure of deoxyribonucleotides, called
deoxynucleotides for short. Each is composed of a base, a sugar, and a
phosphate group. The DNA bases are adenine (A), cytosine (C), guanine

doi:10.1128/9781555817169.ch1 13

9781555816278_013-066_CH01.indd 13 11/5/12 11:42 AM

 14 CHAPTER 1

2 nm (20 Å) (G), and thymine (T), which have either one or two
rings, as shown in Figure 1.2. The bases with two rings
(A and G) are the purines, and those with only one ring
A T (T and C) are pyrimidines. A third pyrimidine, uracil
G C (U), replaces thymine in RNA. The carbons and nitro-
gens making up the rings of the bases are numbered se-
T A
quentially, as shown in the figure. All four DNA bases
T A are attached to the five-carbon sugar deoxyribose. This
1 helical turn = 3.4 nm

sugar is identical to ribose, which is found in RNA, ex-

– 10 bp

Base
cept that it does not have an oxygen attached to the
second carbon, hence the name deoxyribose. The car-
1 helical turn ~

A T
bons in the sugar of a nucleotide are also numbered 1,
T A 2, 3, and so on, but they are labeled with “primes” to
C G distinguish them from the carbons in the bases (Figure
1.2). The nucleotides also have one or more phosphate
A T groups attached to a carbon of the deoxyribose sugar, as
Sugar-phosphate
shown. The carbon to which the phosphate group is at-
tached is indicated, although if the group is attached to
A T
the 5′ carbon (the usual situation), the carbon to which
it is attached is often not stipulated.
G C The components of the deoxynucleotides have spe-
T A cial names. A deoxynucleoside (rather than -tide) is a
base attached to a sugar but lacking a phosphate. With-
T A
Hydrogen 1.2 nm out phosphates, the four deoxynucleosides are called
bond Minor
groove deoxyadenosine, deoxycytidine, deoxyguanosine, and
deoxythymidine. As shown in Figure 1.2, the deoxynu-
A T cleotides have one, two, or three phosphates attached
T A
to the sugar and are known as deoxynucleoside mono-
phosphates, diphosphates, or triphosphates, respectively.
C G The individual deoxynucleoside monophosphates, called
A T deoxyguanosine monophosphate, etc., are often abbre-
2.2 nm viated dGMP, dAMP, dCMP, and dTMP, where the d
Major
groove stands for deoxy; the G, A, C, or T stands for the base;
and the MP stands for monophosphate. In turn, the di-
G C phosphates and triphosphates are abbreviated dGDP,
C G
dADP, dCDP, and dTDP and dGTP, dATP, dCTP, and
dTTP, respectively. The phosphate attached to the sugar
A T is called the α phosphate, while the next two are called
G C the β and γ phosphates, respectively, as shown in the fig-
ure. Collectively, the four deoxynucleoside triphosphates
are often referred to as dNTPs.

T A
The DNA Chain
Phosphodiester bonds join each deoxynucleotide link in
T A
the DNA chain. As shown in Figure 1.3, the phosphate
3’ 5’
attached to the last (5′) carbon of the deoxyribose sugar
Right-handed helix of one nucleotide is attached to the third (3′) carbon of
Figure 1.1 Schematic drawing of the Watson-Crick structure the sugar of the next nucleotide, thus forming one strand
of DNA, showing the helical sugar-phosphate backbones of the of nucleotides connected 5′ to 3′, 5′ to 3′, etc.
two strands held together by hydrogen bonding between the
bases. Also shown are the major and minor grooves and the The 5′ and 3′ Ends
dimensions of the helix.
doi:10.1128/9781555817169.ch1.f1.1 The nucleotides found at the ends of a linear piece of
DNA have properties that are biochemically important
and useful for orienting the DNA strand. At one end

9781555816278_013-066_CH01.indd 14 11/5/12 11:42 AM

 The Bacterial Chromosome: DNA Structure, Replication, and Segregation 15

Bases NH2 O
6 7
5 N N N
1N N HN
8
2 4 N9 N H2N N
N H N H N H
3
Purine Adenine Guanine

O O NH2
4
CH3
3N 5 HN HN N

2 6
O O O
N N N N
1 H H H
Pyrimidine Thymine Uracil Cytosine

Sugars CH2OH CH2OH

5’ OH 5’ OH
O O
4’ 1’ 4’ 1’

3’ 2’ 3’ 2’
OH OH OH
2-Deoxyribose Ribose

Nucleotides NH2 O

N
N NH

O N9 NH2
N1 N
O
–O
P O CH2
CH2OH CH2OH 5’
5’ O
O O–
4’ 1’
4’ 1’
O
3’ 2’
3’ 2’
OH N
O NH
5’ dGMP
–O P O
N NH2
O– N
O O O
3’ dCMP
–O P O P O P O CH2
5’
O
O– O– O–
4’ 1’
γ β α
3’ 2’
OH
5’ dGTP
Figure 1.2 Chemical structures of deoxyribonucleotides, showing the bases and sugars and
how they are assembled into a deoxyribonucleotide. doi:10.1128/9781555817169.ch1.f1.2

9781555816278_013-066_CH01.indd 15 11/5/12 11:42 AM

 16 CHAPTER 1

A
O– O O– O O– O
P CH2 P CH2 P CH2
O O 5’ O O 5’ O O 5’ OH
4 ’ 3’ 4’ 3’ 4’ 3’
O 2’ O 2’ O 2’
1’ 1’ 1’
Base Base Base

Phosphodiester
Deoxyribose bond

5’ 5’ 5’
P P P OH
3’ 3’ 3’

A T C

B 5’ 5’ 5’ 5’ 5’
P P P P P OH
5’ end 3’ 3’ 3’ 3’ 3’ 3’ end

A C G T C

T G C A G

Figure 1.3 (A) Schematic drawing of a DNA 3’ end 3’ 3’ 3’ 3’ 3’ 5’ end

chain showing the 3′-to-5′ attachment of the OH P P P P P
phosphates to the sugars, forming phosphodies-
ter bonds. (B) Two strands of DNA bind at the 5’ 5’ 5’ 5’ 5’
bases in an antiparallel arrangement of the
phosphate-sugar backbones. 5’ 3’
doi:10.1128/9781555817169.ch1.f1.3 3’ 5’

of the DNA chain, a nucleotide will have a phosphate
attached to its 5′ carbon that does not connect it to an-
other nucleotide. This end of the strand is called the 5′
end or the 5′ phosphate end (Figure 1.3B). On the other
end, the last nucleotide lacks a phosphate at its 3′ car-
bon. Because it has only a hydroxyl group (the OH in
Figure 1.3B), this end is called the 3′ end or the 3′ hy-
droxyl end.
Base Pairing
The sugar and phosphate groups of DNA form what is
often called a backbone to support the bases, which jut
out from the chain. This structure allows the bases from
one single strand of DNA to form hydrogen bonds with
another strand of DNA, thereby holding together two
separate nucleotide chains (Figure 1.3B). The first clue
that pairing between specific bases could form the basis
for the structure of DNA came from Erwin Chargaff’s
observation about the ratios of the bases; no matter the
source of the DNA, the concentration of guanine (G) al-
ways equals the concentration of cytosine (C) and the
concentration of adenine (A) always equals the concen-
tration of thymine (T). These ratios, named Chargaff’s
rules, gave Watson and Crick one of the essential clues
to the structure of DNA. They proposed that the two
strands of the DNA are held together by specific hydro-
gen bonding between the bases in opposite strands, as
shown in Figure 1.4. Thus, the amounts of A and T and
of C and G are always the same because A’s pair only
with T’s and G’s pair only with C’s to hold the DNA
strands together. Each A-and-T pair and each G-and-C
pair in DNA is called a complementary base pair, and
the sequences of two strands of DNA are said to be
complementary if one strand always has a T where there
is an A in the other strand and a G where there is a C in
the other strand.
It did not escape the attention of Watson and Crick
that the complementary base-pairing rules essentially ex-
plain heredity. If A pairs only with T and G pairs only
with C, then each strand of DNA can replicate to make
a complementary copy, so that the two replicated DNAs
will be exact copies of each other. Offspring containing

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 The Bacterial Chromosome: DNA Structure, Replication, and Segregation 17

H minor groove. Most of the modifications to DNA that

N N H O CH3 are discussed in this and later chapters occur in the ma-
H C jor groove of the helix.
C C C C
N
Deoxyribose C N H N C H
The Mechanism of DNA Replication
N C C N
The molecular details of DNA replication are probably
H O Deoxyribose
similar in all organisms on Earth. The basic process of
replication involves polymerizing, or linking, the nucle-
Adenine Thymine otides of DNA into long chains, or strands, using the
sequence on the other strand as a guide. Because the nu-
H cleotides must be made before they can be put together
H N O H N H into DNA, the nucleotides are an essential precursor of
C DNA synthesis.
C C C C
N
Deoxyribose C N H N C H Deoxyribonucleotide Precursor Synthesis
N C C N The precursors of DNA synthesis are the four deoxy-
O Deoxyribose
ribonucleoside triphosphates, dATP, dGTP, dCTP, and
N H
dTTP. The triphosphates are synthesized from the cor-
H responding ribose nucleoside diphosphates by the path-
Guanine Cytosine way shown in Figure 1.5. In the first step, the enzyme
Figure 1.4 The two complementary base pairs found in DNA. ribonucleotide reductase reduces (i.e., removes an oxy-
Two hydrogen bonds form in adenine-thymine base pairs. gen from) the ribose sugar to produce the deoxyribose
Three hydrogen bonds form in guanine-cytosine base pairs. sugar by changing the hydroxyl group at the 2′ position
doi:10.1128/9781555817169.ch1.f1.4
(the second carbon) of the sugar to a hydrogen. Then,
an enzyme known as a kinase adds a phosphate to the
the new DNAs would have the same sequence of nucleo- deoxynucleoside diphosphate to make the deoxynucleo-
tides in their DNAs as their parents and thus would be side triphosphate precursor.
exact copies of their parents. The deoxynucleoside triphosphate dTTP is synthe-
sized by a somewhat different pathway from the other
Antiparallel Construction three. The first step is the same. Ribonucleotide reduc-
As mentioned at the beginning of this section, the tase synthesizes the nucleotide dUDP (deoxyuridine
complete DNA molecule consists of two long chains diphosphate) from the ribose UDP. However, from
wrapped around each other in a double helix (Figure then on, the pathway differs. A phosphate is added to
1.1). The double-stranded molecule can be thought of make dUTP, and the dUTP is converted to dUMP by a
as being like a circular staircase, with the alternating
phosphates and deoxyribose sugars forming the railings Figure 1.5 The pathways for synthesis of deoxynucleotides
and the bases connected to each other forming the steps. from ribonucleotides. Some of the enzymes referred to in the
However, the two chains run in opposite orientations, text are identified. THF, tetrahydrofolate; DHF, dihydrofolate.
with the phosphates on one strand attached 5′ to 3′, 5′ doi:10.1128/9781555817169.ch1.f1.5
to 3′, etc., to the sugars and those on the other strand GDP dGDP dGTP
Ribonucleotide Kinase
attached 3′ to 5′, 3′ to 5′, etc. This arrangement is called reductase
antiparallel. In addition to phosphodiester bonds run- CDP dCDP dCTP
ning in opposite directions, the antiparallel construction
causes the 5′ phosphate end of one strand and the 3′ hy- ADP dADP dATP
droxyl end of the other to be on the same end of the UDP dUDP dUTP
double-stranded DNA molecule (Figure 1.3B).
Phosphatase
The Major and Minor Grooves THF THF dUMP
Dihydrofolate Thymidylate synthetase
Because the two strands of DNA are wrapped around
reductase
each other to form a double helix, the helix has two DHF DHF dTMP
grooves between the two strands (Figure 1.1). One of Kinase
these grooves is wider than the other, so it is called the
major groove. The other, narrower groove is called the dTTP

9781555816278_013-066_CH01.indd 17 11/5/12 11:42 AM

 18 CHAPTER 1

A Polymerization reaction β γ β γ
O O O O

P O P O P O P O

O O
α α
O– O O– O O– O
P CH2 P CH2 P CH2
O– O 5’ O O 5’ O O 5’ OH
4’ 3’ 4’ 3’ 4’ 3’
O 2’ O 2’ O 2’
H H H
1’ 1’ 1’
Base Base Base

B Antiparallel strands
O– O O– O O– O
P CH2 P CH2 P CH2
O– O 5’ O O 5’ O O 5’ OH
4’ 3’ 4’ 3’ 4’ 3’
O 2’ O 2’ O 2’
H H H
1’ 1’ 1’
Base Base Base

Base Base Base

1’ 1’ 1’
H H H
2’ O 2’ O 2’ O
4’ 3’ 4’ 3’ 4’ 3’
OH 5’ O O 5’ O O 5’
CH2 P CH2 P CH2
O O– O O–

C Base flipping
O– O O– O O– O
P CH2 P CH2 P CH2
O– O 5’ O O 5’ O O 5’ OH
4’ 3’ 4’ 3’ 4’ 3’
O 2’ O 2’ O 2’
H H H
1’ 1’ 1’
Base Base Base

Base Base
1’ 1’
H H
O 2’ O 2’
4’ 3’ 4’ 3’
5’ OH
O O 5’
2
O
CH H2C CH2
O P
P 5’ O
O 3’ 4’ O–
O– 2’ O
1’
Flipped base Base

Figure 1.6 Features of DNA. (A) Polymerization of the deoxynucleotides during DNA
synthesis. The β and γ phosphates of each deoxynucleoside triphosphate are cleaved off to
provide energy for the polymerization reaction. (B) The strands of DNA are antiparallel. (C) A
single base can be flipped out from the double helix, which could be important in recombina-
tion and repair. doi:10.1128/9781555817169.ch1.f1.6

9781555816278_013-066_CH01.indd 18 11/5/12 11:42 AM