Bacterial and Eukaryotic

DNA
REPLICATION

Overview
A) CHROMOSOME STRUCTURE
B) MODE OF REPLICATION
C)THE REPLICATION PROCESS
D)CONFORMATIONS OF
DNA REPLICATION

Genetic material of Prokaryotic cells

consist of DNA only :

and so should not be

called chromosomes

Bacterial DNA Structure

Bacteria have closed, circular DNA
Genome: genetic material in an
organism
E. coli
4 million base pairs
DNA- 107 long and 20 in diameter
cell2x103 long and 8x104 in
diameter
DNA takes up around 10% of cell volume

Genetic material of Eukaryotic cells consist

of:

DNA
protein

DNA has:
1. negative charges
distributed along
its length

2. positively charged
(basic) proteins called
histones bonded to it

Functions of the histones:

1. organize the chromosome

physically
2. regulate the activities of
the DNA

histon
es

Nearly 2m of DNA are

crammed into each
human cell.
What does this mean?

A great deal of
information can
be stored!!

A closer at DNA:

Four Key elements of DNA structure

1) a doublestranded helix
2) of uniform
diameter
3)

twisting to the
right

4) the two
strands
running in
opposite
directions

Overview
A) CHROMOSOME STRUCTURE
B) MODE OF REPLICATION
C)THE REPLICATION PROCESS
D)CONFORMATIONS OF
DNA REPLICATION

Three possible replication patterns:

1. Semiconservative
replication
2. Conservative replication
3. Dispersive replication

Semiconserv
ative
replication
Conservat
ive
replicatio
n
Dispersi
ve
replicati
on

What do you think is

the acceptable mode
of replication today?

Semiconservative replication
Each parent strand serves as a template for
a new strand and the two new DNA strands
each have one old and one new strand

Parent
strands
New /
daughter
strand

Meselson and Stahl experiment

[1958] demonstrates
semiconservative replication:

N
medium
15

E. coli grown in the presence

of 15N (a heavy isotope of
Nitrogen) for many generations
Cells get heavy-labeled DNA

Sampled
at:
0 min

20
min

40
min

E. coli placed in
medium containing
only 14N (a light
isotope of Nitrogen)

Cells broken open

to extract DNA

4 Suspended DNA in cesium

chloride (CsCl) solution.

0 min

20 min

CsCl density gradient

centrifugation

40 min

DNA
14

Both
strands
heavy

F1
generation
DNA (one
heavy/one
light strand)

F2 generation
DNA:
Two light
strands
(one heavy/one
light strand)

15

Overview
A) CHROMOSOME STRUCTURE
B) SEMICONSERVATIVE
REPLICATION
C)THE REPLICATION PROCESS
D)CONFORMATIONS OF
DNA REPLICATION

Central dogma
replication transcripti
on

DNA

translati
on

protein

RNA

reverse
transcriptio
n

22

What is the
significance of
DNA replication?

 For the transfer of genetic

information with high
fidelity
-from cell to cell
-from one generation
to another

FOUR requirements for DNA to replicate

1. DNA to act as
a template for
complementary
base pairing.

2. The four
deoxyribonucleoside
triphosphates:
dATP, dGTP, dCTP &
dTTP.

3. A source of chemical energy is

needed to drive this highly
endergonic
reaction.
The nucleotides
arrive as nucleosides
DNA bases with PPP
P-P-P = energy for bonding
DNA bases arrive with their own
energy source for bonding

dATP

dGTP

dTTP

dCTP

4. Enzymes that
catalyzes the
reactions

DNA
Polymerase III

Energy of Replication
Where does energy for bonding
usually come from?
We come
with our own
energy!

You
remember
ATP!
Are
Are there
there
other
other ways
energy
to
get energy
nucleotides?
out
You of
betit?
!

energy
energy

ATP
TTP
GTP
CTP

And we
leave behind a
nucleotide!

modified nucleotide

TMP
AMP
GMP
CMP
ADP

Basic rules of DNA replication

Specificity of base
pairing
Synthesis direction 53 (C at 3 position in
3 end has free OH)
Nucleotides added at
3 of growing strand
DNA polymerase

Components of DNA Replication

DNA polymerases- Deoxynucleotide polymerization
Helicase -Processive unwinding of DNA
Topoisomerases Relieve torsional strain that results from
helicase-induced unwinding
RNA primase Initiates synthesis of RNA primers
Single-strand binding proteins Prevent premature
reannealing of dsDNA
DNA ligase Seals the single strand nick between the
nascent chain and Okazaki fragments on lagging strand

DNA replication occurs in two steps:

1.DNA is locally
denatured
WHY?
(unwound)
To separate the
two template
strands and
make them
available for
base pairing.

Unzipping
of DNA

What are the bonds

present in DNA?

2.

The new
nucleotides
are linked by
covalent
bonding to
each growing
strand in a
sequence
determined
by
complementar

Bacterial DNA
Replication

Enzymes

Three Stages of replication

1) Initiation
occurs at the origin of replication

2) Elongation
involves the addition of new
nucleotides based on
complementarity of the template
strand

3) Termination
occurs at a specific termination site

Initiation
Each bacterial DNA
has only one origin of
replication

 Unwind DNA
helicase enzyme
unwinds part of DNA helix
stabilized by single-stranded binding
proteins
helicase

gle-stranded binding proteins

replication fork

A primer is :
- required to
start DNA
replication
a short
single strand
of RNA.
- synthesised
by primase.

Many Proteins at the

Replication Fork

Elongation
Once priming is complete DNA
pol III is loaded into the DNA
and replication begins
Nucleophilic attack by the 3' OH
on the alpha phosphate
releases pyrophosphate
Subsequently hydrolyzed (by
inorganic phosphatase) into two
phosphates
This hydrolysis drives DNA
synthesis to completion

REMEMBER:
Nucleotides are always added to
the growing strand at the 3 end
the end at which the DNA strand
has a free OH group on the 3
carbon of its terminal deoxyribose

Protein complexes of the replication

fork

Bacterial DNA
Polymerase

How are Okazaki fragments linked?

Each Okazaki
fragment
requires a
primer.

The final
phosphodiester linkage
between fragments is
catalyzed by DNA ligase.

Termination

In some DNA
molecules, specific
termination sequences
block further replication.
A termination protein,
called Tus in E. coli, binds
to these sequences. Tus
blocks the movement of
helicase, thus stalling the
replication fork and
preventing further DNA
replication.

In some DNA
molecules, replication
is terminated whenever
two replication forks
meet.

Eukaryotic DNA
Replication

Initiation
The eukaryotic origins are shorter
than that of E. coli.
Requires DNA-pol (primase
activity) and DNA-pol (polymerase
activity and helicase activity).
Needs topoisomerase and replication
factors (RF) to assist.

52

Elongation
DNA replication and nucleosome
assembling occur simultaneously.
Overall replication speed is
compatible with that of prokaryotes.

53

DNA Polymerase in Eukayotes

Termination
3'
5'

5'
3'

3'

5'

5'

3'

3'
5'

connection of discontinuous
segment
5'
3'

3'

5'

5'

3'
55

A eukaryotic chromosome
May have hundreds or even
thousands of replication origins
DNA is
replicated
simultaneously
at the origins.

Proofreading procedure
DNA replication is
not perfect
1) the high speed of replication
- (1000 nucleotides per second)
2) spontaneous chemical flip-flops in the
bases

) occasionally DNA polymerase

incorporates incorrectly matched bases

Errors in Replication
For Bacterial cells
-error is at a frequency of 1 in 109
to1010 nucleotides
For Eukaryotic cells
-error is expected to be one every
115.7 days to 1157.4 days

 A much higher rate of error in

replication is incorporated in
exposure to various
mutagens (e.g. X-ray)

Editing & proofreading DNA

1000 bases/second
DNA polymerase I
proofreads & corrects
mistakes
repairs mismatched
bases
removes abnormal
bases
repairs damage
throughout life

reduces error rate

from
1 in 10,000 to
1 in 100 million bases

Fast & accurate!

It takes E. coli <1 hour to copy
5 million base pairs in its single
chromosome
divide to form two identical
daughter cells

Human cell copies its 6 billion

bases & divide into daughter cells
in only few hours
remarkably accurate
only ~1 error per 100 million bases
~30 errors per cell cycle

Overview
A) CHROMOSOME STRUCTURE
B) MODE OF REPLICATION
C)THE REPLICATION PROCESS
D)CONFORMATIONS OF
DNA REPLICATION

CONFORMATIONS OF DNA
REPLICATION
LINEAR DNA REPLICATION
CIRCULAR DNA :THETA
CONFORMATION
CIRCULAR DNA :ROLLING CIRCLE
CONFORMATION

Conformations of
Replication

THE
END