SMBM 201:

CELL BIOLOGY AND GENETICS

The molecular basis of life: DNA- as a carrier of

genetic information; replication; transcription;
translation and their regulation.
Why did DNA evolve as genetic material?

• RNA molecules can perform the cellular

functions that are carried out by DNA.
• eg. Many viruses contain RNA as the genetic
material.
• DNA is more stable than RNA.
 The central dogma of life
• The biological information flows from DNA to
RNA, and from there to proteins. This is the
central dogma of life.
• As the carrier of genetic information, DNA in a
cell must be duplicated (replicated)
maintained and passed down accurately to the
daughter cells.
In DNA there are four bases: adenine (A), guanine
(G), thymine (T) and cytosine (C). Adenine and
guanine are purines; thymine and cytosine are
pyrimidines.

A nucleoside is a pyrimidine or purine base

covalently bonded to a sugar. In DNA, the sugar
is deoxyribose and so this is a deoxynucleoside.
There are four types of deoxynucleoside in DNA;
deoxyadenosine,deoxyguanosine,
deoxythymidine and deoxycytidine.
A nucleotide is base + sugar + phosphate
covalently bonded together. In DNA, where the
sugar is deoxyribose, this unit is a
deoxynucleotide.
In DNA the nucleotides are covalently joined together by 3’5’
phosphodiester bonds to form a repetitive sugar–phosphate
chain which is the backbone to which the bases are attached.

3′5′ phosphodiester bonds formed

between nucleotides in a DNA molecule.
The DNA sequence is the sequence of A, C, G and
T along the DNA molecule which carries the
genetic information.
For example,
ACTTTCAGACC is part of the base sequence of one
gene and codes for part of one protein whereas
TGGAACCGTCA is part of the base sequence of a
different gene coding for a different protein.
 Overview: Life’s Operating Instructions
• 1953
– James Watson and
Francis Crick
– Structure of the
molecule of
inheritance
– Deoxyribonucleic acid

• Rosalind Franklin
– Used X-ray
crystalography to
take the first picture
of the molecule

© 2011 Pearson Education, Inc.

DNA is composed of two strands wound
round each other to form a double helix,
with the bases on the inside and the sugar–
phosphate backbones on the outside. In the
double helix , the two DNA strands are
organized in an antiparallel arrangement
(i.e. the two strands run in opposite
directions, one strand is orientated 5’→3’
and the other is orientated 3’→5’). The
bases of the two strands form hydrogen
bonds to each other; A pairs with T and G
pairs with C. This is called complementary
base pairing .
 Three possible models of DNA replication
Semi-conservative mechanism: During replication, the strands of the double
helix separate and each acts as a template to direct the synthesis of a
complementary daughter strand using deoxyribonucleoside 5‘ –triphosphate as
precursors. Thus, each daughter cell receives one of the original parental
strands.

Conservative replication: The whole original double helix acts as a template

for a new one, one daughter molecule would consist of the original parental
DNA, and the other daughter would be totally new DNA.

Dispersive replication: In this model, the parental double helix is broken into
double-stranded DNA segments and just like conservative mode of replication
acts as templates for the synthesis of new double-stranded DNA segments. The
segments then reassemble into complete DNA double helices, each with
Alternative models of DNA replication :
The Meselson-Stahl experiment, which showed that DNA replicates
semiconservatively
1. Meselson and Stahl (1958) grew E. coli in a heavy) isotope of
nitrogen, 15
N in the form of 15
NH4Cl. Because it is heavier, DNA
containing 15
N is more dense than DNA with normal 14
N, and so can
be separated by CsCl density gradient centrifugation.

2. Once the E. coli were labeled with heavy 15N, the researchers shifted
the cells to medium containing normal 14
N, and took samples at time
points. DNA was extracted from each sample and analyzed in CsCl
density gradients.

3. After one replication cycle in normal 14N medium, all DNA had density
intermediate between heavy and normal. After two replication cycles,
there were two bands in the density gradient, one at the intermediate
position, and one at the position for DNA containing entirely 14N.
Results compared with the three proposed models:
a. Does not fit conservative model, because after one
generation there is a single intermediate band, rather than
one with entirely 15N DNA and another with entirely 14N DNA.
b. The dispersive model predicted that a single band of DNA
of intermediate density would be present in each
generation, gradually becoming less dense as increasing
amounts of 14N were incorporated with each round of
replication. Instead, Meselson and Stahl observed two
bands of DNA, with the intermediate form decreasing over
time.
c. The semiconservative model fits the data very well.
 DNA REPLICATION IN BACTERIA

DNA polymerases DNA polymerase I from E. coli catalyzes the stepwise

addition of deoxyribonucleotides to the 3’-OH end of a DNA chain:
(DNA)n residues + dNTP → (DNA)n+1 + PPi
The enzyme has the following requirements:
● all four dNTPs (dATP, dGTP, dTTP and dCTP) must be present to be
used as precursors; Mg2+ is also required;
● a DNA template is essential, to be copied by the DNA polymerase;
● a primer with a free 3’-OH that the enzyme can extend.
• DNA polymerase I is a template-directed enzyme, that
is it recognizes the next nucleotide on the DNA
template and then adds a complementary nucleotide
to the 3’-OH of the primer, creating a 3’5’
phosphodiester bond, and releasing pyrophosphate.
It involves nucleophilic attack of the 3’-OH of the
primer on the α-phosphate group of the incoming
nucleotide. The primer is extended in a 5’ → 3’
direction.
• DNA polymerase I also corrects mistakes in DNA by

removing mismatched nucleotides (i.e. it has proof-

reading activity). Thus, during polymerization, if the

nucleotide that has just been incorporated is incorrect

(mismatched), it is removed using a 3’ → 5’

exonuclease activity. This gives very high fidelity; an

error rate of less than 10–8 per base pair.

DNA polymerase also has a 5’ → 3’exonuclease activity; it can hydrolyze nucleic acid

starting from the 5’ end of a chain. This activity plays a key role in removing the RNA

primer used during replication . Thus, overall, DNA polymerase I has three different

active sites on its single polypeptide chain; 5’ → 3’ polymerase, 3’ → 5’ exonuclease

and 5’ → 3’ exonuclease. As well as its role in DNA replication, DNA

polymerase I is involved in DNA repair, for example removing UV-induced

alterations such as pyrimidine dimers.

E. coli also contains two other DNA polymerases, DNA polymerase II and DNA

polymerase III. As with DNA polymerase I, these enzymes also catalyze the

template-directed synthesis of DNA from deoxynucleotidyl 5’-triphosphates, need

a primer with a free 3’-OH group, synthesize DNA in the 5’ → 3’ direction, and

have 3’ → 5’ exonuclease activity. Neither enzyme has 5’ → 3’ exonuclease activity

Double-stranded DNA is antiparallel ; one strand runs 5’ → 3’ and the

complementary strand runs 3’ → 5’.

New DNA to be made 5’ → 3’ for one daughter strand and 3’ → 5’ for the

other daughter strand. However, all DNA polymerases make DNA only in the

5’ → 3’ direction and never in the 3’ → 5’ direction.

What actually happens is that on the template strand with 3’ →

5’orientation?

New DNA is made in a continuous piece in the correct 5’ → 3’

direction. This new DNA is called the leading strand .

On the other template strand (that has a 5’ → 3’ orientation), DNA

polymerase synthesizes short pieces of new DNA (about 1000–2000

nucleotides long) in the 5’ → 3’ direction and then joins these pieces

together. The small fragments are called Okazaki fragments after their

discoverer. The new DNA strand which is made by this discontinuous

RNA primer
DNA polymerase cannot start DNA synthesis without a primer.
Even on the lagging stand, each Okazaki fragment requires an
RNA primer before DNA synthesis can start. The primer used in
each case is a short piece of RNA and is synthesized by an RNA
polymerase called primase . Primase can make RNA directly on
the single-stranded DNA template because, like all RNA
polymerases, it does not require a primer to begin synthesis. The
RNA primer made by primase is then extended by DNA
polymerase III . DNA polymerase III synthesizes DNA for both the
leading and lagging strand. After DNA synthesis by DNA
polymerase III, DNA polymerase I uses its 5’ → 3’ exonuclease
activity to remove the RNA primer and then fills the gap with new
DNA . DNA polymerase III cannot carry out this task because it
lacks the 5’ → 3’ activity of DNA polymerase I. Finally, DNA ligase
joins the ends of the DNA fragments together .
Fig. 3.5 Model for the formation of a replication bubble at a replication origin in
E. coli and the initiation of the new DNA strand
 Figure 16.13

Primase

3
Topoisomerase
5 RNA
3 primer
5
3

Helicase

5
Single-strand binding
proteins
Fig. 3.6c-e Model for the events occurring around a single replication
fork of the
E. coli chromosome
1. When DNA denatures at the oriC, replication forks are
formed. DNA replication is usually bi-directional, but will
consider events at just one replication fork (Figure 3.6):
a. Single-strand DNA-binding proteins (SSBs) bind the
ssDNA formed by helicase, preventing reannealing.
b. Primase synthesizes a primer on each template strand.
c. DNA polymerase III adds nucleotides to the 3’ end of
the primer, synthesizing a new strand complementary to
the template, and displacing the SSBs. DNA is made in
opposite directions on the two template strands.
d. New strand made 5’ → 3’ in same direction as
movement of the replication fork is leading strand, while
new strand made in opposite direction is lagging strand.
Leading strand needs only one primer, while lagging
needs a series of primers.
2. Helicase denaturing DNA causes tighter winding in other
parts of the circular chromosome. Gyrase relieves this
tension.
3. Leading strand is synthesized continuously, while lagging
strand is synthesized discontinuously, in the form of
Okazaki fragments. DNA replication is therefore
semidiscontinuous.
4. Each fragment requires a primer to begin, and is extended
by DNA polymerase III.
5. Okazaki data show that these fragments are gradually
joined together to make a full-length dsDNA chromosome.
DNA polymerase I uses the 3’-OH of the adjacent DNA
fragment as a primer, and simultaneously removes the RNA
primer while resynthesizing the primer region in the form
of DNA. The nick remaining between the two fragments is
sealed with DNA ligase.
 DNA REPLICATION IN EUKARYOTES

Cell cycle
The life of a eukaryotic cell can be defined as a
cell cycle . Mitosis and cell
division occur in the M phase which lasts for
only about 1 h. This is followed by the G 1 phase
(G for gap), then the S phase (S for synthesis),
during which time the chromosomal DNA is
replicated, and finally the G2 phase in which the
cells prepare for mitosis.
Replication of each linear DNA molecule in a chromosome
starts at many origins, one every 30–300 kb of DNA
depending on the species and tissue, and proceeds
bi-directionally from each origin. The use of multiple
origins is essential in order to ensure that the large
amount of chromosomal DNA in a eukaryotic cell is
replicated within the necessary time period. At each
origin, a replication bubble forms consisting of two
replication forks moving in opposite directions.
The DNA replicated under the control of a single origin is
called a replicon.
Eukaryotic enzymes:

Five common DNA polymerases from mammals.

1. Polymerase  (alpha): nuclear, DNA replication, no proofreading

2. Polymerase  (beta): nuclear, DNA repair, no proofreading

3. Polymerase  (gamma): mitochondria, DNA repl., proofreading

4. Polymerase  (delta): nuclear, DNA replication, proofreading

5. Polymerase  (epsilon): nuclear, DNA repair (?), proofreading

Leading and lagging strands
The RNA primers required are made by DNA polymerase 
which carries a primase subunit. DNA polymerase α initiates
synthesis of the lagging strand, making first the RNA primer
and then extending it with a short region of DNA. DNA
polymerase δ then synthesizes
the rest of the Okazaki fragment. The leading strand is
synthesized by DNA polymerase δ. The δ enzyme has 3’ → 5’
exonuclease activity and so can proofread the DNA made,
but DNA polymerase α has no such activity.
Each telomere contains many copies of a repeated hexanucleotide sequence
that is G-rich; in Tetrahymena it is GGGTTG. Telomerase carries, as an integral
part of its structure, an RNA molecule, part of which is complementary to this
G-rich sequence. The RNA molecule of telomerase is envisaged to
hydrogen-bond to the telomere end. Then, using the RNA as a template,
telomerase copies the RNA template (hence this enzyme is a reverse
transcriptase and adds six deoxynucleotides to the telomere DNA end.
Telomerase then dissociates from the DNA, re-binds at the new telomere end
and repeats the extension process. It can do this hundreds of times before
finally dissociating. The newly extended DNA strand can then act as a
template for normal DNA replication (lagging strand synthesis by DNA
polymerase α) to form double-stranded chromosomal DNA.