256 Biochemistry, Molecular Biology, and Genetics

FIGURE 17-8 Three modified nucleosides found in most transfer RNAs.

a. In eukaryotic cells, many nucleotides in tRNA are modified. Modified nucleotides con-
taining pseudouridine (Y), dihydrouridine (D), and ribothymidine (T) are present in most
tRNAs (Figure 17-8).
b. All tRNA molecules have a similar cloverleaf structure even though their base sequen-
ces differ (Figure 17-9).
(1) The first loop from the 50 end, the D loop, contains dihydrouridine.
(2) The middle loop contains the anticodon, which base pairs with the codon in mRNA.
(3) The third loop, the TYC loop, contains both ribothymidine and pseudouridine.
(4) The CCA sequence at the 30 end carries the amino acid.

II. SYNTHESIS OF DNA (REPLICATION)

A. Mechanism of replication
1. Replication is bidirectional and semiconservative (Figure 17-10).
a. Bidirectional means that replication begins at a site of origin and simultaneously
moves out in both directions from this point.
(1) Prokaryotes have one site of origin on each chromosome.
(2) Eukaryotes have multiple sites of origin on each chromosome.
b. Semiconservative means that, following replication, each daughter molecule of DNA
contains one intact parental strand and one newly synthesized strand joined by base
pairs.

FIGURE 17-9 The cloverleaf structure of transfer RNA.

Bases that commonly occur in a particular position are indi-
cated by letters. Base pairing in stem regions is indicated
by dashed lines between strands. Y, pseudouridine, T, ribo-
thymidine; D, dihydrouridine.
 Chapter 17 DNA Replication and Transcription 257

O=origin
O O

Replication
forks
O O

O O

O O
FIGURE 17-10 Replication of a eukaryotic chromosome. Solid
lines are parental strands. Dotted lines are newly synthesized +
strands. Synthesis is bidirectional from each point of origin (O).

2. Replication forks are the sites at which DNA synthesis is occurring.

a. The parental strands of DNA separate, and the helix unwinds ahead of a replication fork
(Figure 17-11).
b. Helicases unwind the helix, and single-strand binding proteins hold it in a single-
stranded conformation.
c. Topoisomerases act to prevent the extreme supercoiling of the parental helix that
would result as a consequence of unwinding at a replication fork.
d. Topoisomerases break and rejoin DNA chains.

CLINICAL The cancer drug etoposide (VP-16) inhibits topoisomerase and is widely used in
CORRELATES the treatment of lung, ovarian, testicular, and prostate cancer.

e. DNA gyrase, a topoisomerase, is found only in prokaryotes.

CLINICAL Quinolone antibiotics, such as ciprofloxacin, inhibit DNA gyrase and are used
CORRELATES for numerous infections, including complicated urinary tract infections and
lower respiratory tract infections.

3. DNA polymerases catalyze the synthesis of DNA.

a. Prokaryotes have three DNA polymerases: pol I, pol II, and pol III. Pol III is the major
replicative enzyme, and pol I is involved in both DNA replication and repair.
b. Eukaryotes have at least five major species of DNA polymerase: a, b, g, d, and e.
(1) DNA polymerase a is involved in replication of nuclear DNA.
(2) Polymerase d acts in conjunction with a during replication.
(3) Polymerases b and e are involved in repair of nuclear DNA, and g functions in
mitochondria.
c. DNA polymerases can only copy a DNA template in the 30 to 50 direction and produce
the newly synthesized strand in the 50 to 30 direction.
258 Biochemistry, Molecular Biology, and Genetics

Direction of replication fork

5'

3' Parental strand

Lagging strand
DNA polymerase δ
DNA polymerase α
primase topoisomerase

3'

5'
helicase
Parental strand
Leading strand FIGURE 17-11 The eukaryotic rep-
lication complex located at a repli-
DNA polymerase δ cation fork. The lagging strand
5' loops around the complex. Single-
strand binding proteins (not
Parental strand = RNA primer shown) are attached to the regions
3' = New DNA strand of single-stranded DNA.

d. Deoxyribonucleoside triphosphates (dATP, dGTP, dTTP, and dCTP) are the precursors
for DNA synthesis.
(1) Each precursor pairs with the corresponding base on the template strand and forms a
phosphodiester bond with the hydroxyl group on the 30 -carbon of the sugar at the end
of the growing chain (Figure 17-12).

CLINICAL Many of the antiviral drugs used in the treatment of human immunodeficiency
CORRELATES virus (HIV) are analogs of deoxyribonucleoside triphosphates. For instance, the
drug zidovudine (AZT, ZDV) is an analog of thymidine, which lacks the 30 -hydroxyl for the addition of
the next nucleotide, thereby inhibiting the viral DNA polymerase. Dideoxyinosine (ddI) and
zalcitabine (ddC) are similar agents used to treat HIV.

(2) Pyrophosphate is produced and cleaved to two inorganic phosphates.

4. DNA polymerase requires a primer (Figure 17-13).
a. DNA polymerases cannot initiate synthesis of new strands.
b. RNA serves as the primer for DNA polymerase in vivo. The RNA primer, which contains
about 10 nucleotides, is formed by copying of the parental strand in a reaction cata-
lyzed by primase.
c. DNA polymerase adds deoxyribonucleotides to the 30 -hydroxyls of the RNA primers
and subsequently to the ends of the growing DNA strands.
d. DNA parental (template) strands are copied simultaneously at replication forks,
although they run in opposite directions.
(1) The leading strand is formed by continuous copying of the parental strand that runs 30
to 50 toward the replication fork.
(2) The lagging strand is formed by discontinuous copying of the parental strand that runs
30 to 50 away from the replication fork.
l As more of the helix is unwound, synthesis of the lagging strand begins from another
primer. The short fragments formed by this process are known as Okazaki fragments.
l The RNA primers are removed by nucleases (e.g., RNase H), and then the resulting
gaps are filled with the appropriate deoxyribonucleotides by another DNA polymerase.
l Finally, the Okazaki fragments are joined by DNA ligase, an enzyme that catalyzes
formation of phosphodiester bonds between two polynucleotide chains.
 Chapter 17 DNA Replication and Transcription 259

Growing Parental
chain strand
5' 5'
3' 3'
T A T A

T A T A

G C G C
+

C G C G
3'
OH C G C

G T OH T
3'
OH A A
dGTP
G G
5' 5'

FIGURE 17-12 The action of DNA

polymerase. dGTP, deoxyguanosine or Phosphate Deoxyribose Pyrophosphate
triphosphate. groups

e. In eukaryotic cells, about 200 deoxyribonucleotides are added to the lagging strand in
each round of synthesis, whereas in prokaryotes, 1000 to 2000 are added.
5. The fidelity of replication is very high, with an overall error rate of 10–9 to 10–10.
a. Errors (insertion of an inappropriate nucleotide) that occur during replication can be
corrected by editing during the replication process. This proofreading function is per-
formed by a 30 to 50 exonuclease activity associated with the polymerase complex.
b. Postreplication repair processes (e.g., mismatch repair) also increase the fidelity of
replication.

B. Mutations
1. Changes in DNA molecules cause mutations. After replication, these changes result in a per-
manent alteration of the base sequence in the daughter DNA.
2. Changes causing mutations include:
a. Uncorrected errors made during replication
b. Damage that occurs to replicating or nonreplicating DNA caused by oxidative deami-
nation, radiation, or chemicals, resulting in cleavage of DNA strands or chemical alter-
ation or removal of bases
3. Types of mutations include:
a. Point mutations (substitution of one base for another)
b. Insertions (addition of one or more nucleotides within a DNA sequence)
c. Deletions (removal of one or more nucleotides from a DNA sequence)

C. DNA repair (Figure 17-14)

1. In general, repair involves three steps:
a. Removal of the segment of DNA that contains a damaged region or mismatched bases
b. Filling in the gap by action of a DNA polymerase that uses the undamaged sister strand
as a template
c. Ligation of the newly synthesized segment to the remainder of the chain
2. Endonucleases, exonucleases, a DNA polymerase, and a ligase are required for repair.
3. Nucleotide excision repair involves the removal of a group of nucleotides (including the dam-
aged nucleotide) from a DNA strand.
260 Biochemistry, Molecular Biology, and Genetics

5' 3' 5' 3'

1
3'
RNA
primers
First round
of synthesis ( 1 )

3' 5'

Unwinding of parental
strands and second
round of synthesis ( 2 )

5' 3' 5' 3'

Lagging 1 Leading
5'
strand 3'
strand
2 3'

Okazaki 5'
fragments

3' 5'

Removal of
RNA primers

5' 3' 5' 3'

5' Gap
3' 3'

5'

3' 5'

Gap filling by a repair

DNA polymerase

5' 3' 5' 3' 5' 3' Ligation 5' 3'

5' of chains
3'
3' 3'
Joining of FIGURE 17-13 Mechanism of DNA synthesis at the replication
5' 5' fork. Two rounds of polymerase action are shown (Œ and ).
Okazaki fragments The number of nucleotides added in each round is much larger
by ligase than shown; in eukaryotes, about 10 ribonucleotides and 200
deoxyribonucleotides are polymerized on the lagging strand.
3' 5' 3' 5'
Synthesis on the leading strand is continuous. The unshaded
2 polynucleotide regions of the arrows indicate the nucleotides added by the
chains repair action of a DNA polymerase.

4. Base excision repair involves a specific glycosylase that removes a damaged base by hydrolyz-
ing an N-glycosidic bond, producing an apurinic or apyrimidinic site, which is cleaved and,
subsequently, repaired.
5. Mismatch repair involves the removal of the portion of the newly synthesized strand of
recently replicated DNA that contains a pair of mismatched bases.
a. Bacteria recognize the newly synthesized strand because, in contrast to the parental
strand, it has not yet been methylated.
b. The recognition mechanism in eukaryotes is not known.
 Chapter 17 DNA Replication and Transcription 261

Normal DNA

Damage
to bases

glycosylase

N
u
B c
a l
s incision endonuclease e
e o
t
e i
x d
e
c
i e
s x
i excision c
o endonuclease i
n s
Gap i
r o
n
e
p r
a DNA polymerase e
i p
Nick
r a
i
r

DNA ligase

FIGURE 17-14 Base excision and nucleotide excision repair of

DNA. Circles indicate normal bases; and indicate dam-
aged bases. The actual number of nucleotides removed (the size
of the gap) is larger than that shown. Repaired (normal) DNA

D. Rearrangements of genes
1. Several processes produce new combinations of genes, thus promoting genetic diversity.
2. Recombination occurs between homologous DNA segments, that is, those that have very simi-
lar sequences.
3. Transposition involves movement of a DNA segment from one site to a nonhomologous site. Trans-
posons (‘‘jumping genes’’) are mobile genetic elements that facilitate the movement of genes.

CLINICAL Transposons in bacteria are believed to mediate the transfer of antibiotic

CORRELATES resistance between bacteria. The creation of multidrug-resistant organisms is a
growing health concern worldwide.

E. Reverse transcription
1. Synthesis of DNA from an RNA template is catalyzed by reverse transcriptase.
2. Retroviruses contain RNA as their genetic material.
a. The retroviral RNA serves as a template for synthesis of DNA by reverse transcriptase.
b. The DNA that is generated can be inserted into the genome (chromosomes) of the host
cell and be expressed.